Method for detecting cells

ABSTRACT

The present invention relates to methods for detecting the chromatin state of a cell based on recording a super resolution image of nucleosome organization and correlating said imaged with size of nucleosomal clutches, nucleosomal density and/or number of nucleosomes per nucleosomal clutches. Additionally, the invention relates to a kit comprising a first antibody capable of specifically binding to a histone protein and a photo switchable fluorophore linked-secondary antibody and the use of the kit of the invention for detecting the chromatin state of a cell and isolating a cell in an open chromatin state or in a close chromatin state. The invention also relates to a device adapted to detect the chromatin state of a cell.

TECHNICAL FIELD OF INVENTION

The present invention belongs to the field of methods for cellidentification.

BACKGROUND OF INVENTION

Pluripotent stem cells have potential to differentiate into any of thethree germ layers: endoderm, mesoderm, or ectoderm and provide a chanceto obtain a renewable source of healthy cells and tissues to treat awide array of diseases.

Methods currently used to detect/isolate pluripotent cells have inherentexperimental variability and low efficiency, and are (1) mechanicalisolation based on morphology that requires experience, and is laboriousand not efficient; (2) quantification of the endogenous expression ofstem cell transcription factors (OCT4, SOX2, etc.) in live cells, whichrequires genome modification; (3) fluorescence-activated cell sorting(FACS)-based analysis using cell surface markers (SSEA-4, TRA-1-60,etc.), which requires use of antibody based staining that is inherentlyvariable; and (4) more recently, a pluripotent stem cell-specificadhesion signature, which is dependent on the surface properties of cellclusters and thus interrogates the population and not individual cells.Additionally, the identification of high-grade pluripotent hiPSCs istime consuming, requiring the generation of teratomas and severaladditional pluripotency test.

Several studies of chromosome territory occupation and genomedistribution inside the nucleus show that the epigenome is dynamic and,that among other processes; it contributes to gene expression and celldifferentiation.

Recent studies have revealed key differences in chromatin states ofpluripotent cells as compared to differentiated cell types.

The spatial organization of chromatin inside the nucleus plays a keyfunctional role. However, how nucleosomes are arranged to form thechromatin fiber is still highly debated.

The existence of a hierarchical organization of the chromatin fiberinside intact eukaryotic nuclei in vivo has recently been debated aftercryo-electron microscopy, small-angle X-ray scattering (SAXS) andelectron spectroscopic imaging experiments failed to detect the 30-nmfiber. The structural information obtained in these studies led to theoverall conclusion that the eukaryotic nuclei are mainly composed of 10nm fibers even though the core histone proteins could not be identifiedunequivocally using these methods due to their lack of molecularspecificity. In addition, genome-wide analyses have revealed thatnucleosomes are depleted at promoter and terminator regions and at manyenhancers and that nucleosomes occupy preferred positions in genes andnon-gene regions. Since the 30-nm fiber arrangement imposes specificconstrains on nucleosome occupancy and positioning, these genome-wideanalyses along with the latest imaging results argue against ahierarchical organization of nucleosomes along the chromatin fiber.

Conventional microscopy have shown that heterochromatin appears in largeregions in pluripotent cells but it was confined to small foci indifferentiated cells, confirming that chromatin in pluripotent cellsassumes a globally more open conformation (Meshorer E. et al., 2006).

Up to date, however, the super-resolution studies of DNA and histoneshave not addressed questions regarding the organization of single orgroups of nucleosomes, the overall nucleosome occupancy level of DNA andwhether these parameters are consistent with the 30 nm fiber model ofchromatin. How the chromatin organization changes at the nanoscale levelas a function of cell state such as pluripotency and differentiation,while of fundamental importance, has also not been studied. In general,what has been lacking is a quantitative approach that can count anddetermine the number of nucleosomes within the chromatin fiber and thusidentify nucleosome spatial arrangement at the nanoscale level.

Given the current debate on nucleosome occupancy, positioning andorganization, and the importance of these parameters for DNAaccessibility and gene expression, novel methods that allow quantitativevisualization of nucleosome organization with high molecular specificityat the nanometer length scales in individual intact nuclei and leadingto determine the chromatin state of a cell without the disadvantages ofharsh sample preparation, lack of molecular specificity or low spatialresolution are needed.

SUMMARY OF THE INVENTION

Combining quantitative super-resolution nanoscopy with computersimulations the inventors detected a striking heterogeneity in thenucleosome organization of intact eukaryotic nuclei. Nucleosomes formedgroups of varying sizes, which they term “clutches” and these wereinterspersed with nucleosome-depleted regions. Remarkably, the mediannumber of nucleosomes and their compaction inside clutches highlycorrelated with cellular state, such that clutch size is predictive ofpluripotency grade. Ground-state pluripotent stem cells had, on average,less dense clutches containing fewer nucleosomes. RNA polymerase IIpreferentially associated with the smallest clutches. These resultsprovide novel insights into chromatin organization at the nanoscalelevel and open new possibilities for identification of stem cellsthrough the structural organization of their chromatin fibers.

In a first aspect, the invention relates to a method for detecting thechromatin state of a cell comprising

-   -   a) contacting a sample containing cells with a first antibody        capable of specifically binding to a histone protein,    -   b) contacting the antibody:histone complex formed in step a)        with a secondary antibody having at least one photoswitchable        fluorophore adapted to be optically excited at a certain        wavelength Δ₁ and to emit light at a wavelength λ₂ different        from λ₁,    -   c) recording a super resolution image of nucleosome organization        by means of a sensor being sensitive at least to the wavelength        of emission of the photoswitchable fluorophore by exciting the        sample with an optical radiation having a wavelength λ₁,    -   d) correlating the image obtained in step c) with size of        nucleosomal clutches, nucleosomal density and/or number of        nucleosomes per nucleosomal clutches, and    -   e) comparing data obtained in step d) with a corresponding        reference value to obtain a score based on size of nucleosomal        clutches, nucleosomal density and/or number of nucleosomes per        nucleosomal clutch,

wherein if the cell comprises smaller clutches, less densely compactednucleosomes or less nucleosomes per clutches compared to thecorresponding reference value is indicative that said cell is in an openchromatin state and wherein if the cell comprises bigger clutches, moredensely compacted nucleosomes or more nucleosomes per clutches comparedto the corresponding reference value is indicative that said cell is ina close chromatin state.

In a second aspect, the invention relates to a method for isolating acell in an open chromatin state comprising

-   -   a) detecting the chromatin state of a cell by a method according        to the invention, and    -   b) isolating a cell having smaller clutches, less densely        compacted nucleosomes or less nucleosomes per clutches.

In a third aspect, the invention relates to a method for isolating acell in a close chromatin state comprising

-   -   a) detecting the chromatin state of a cell by a method according        to the invention, and    -   b) isolating a cell having bigger clutches, more densely        compacted nucleosomes or more nucleosomes per clutches

In a fourth aspect, the invention relates to a kit comprising a firstantibody capable of specifically binding to a histone protein and aphotoswitchable fluorophore linked-secondary antibody.

In a fifth aspect, the invention relates to the use of a kit of theinvention for detecting and isolating a cell in an open chromatin stateor in a close chromatin state.

In a sixth aspect, the invention relates to a device adapted to detectthe chromatin state of a cell comprising

-   -   a source of optical radiation adapted to emit light at a        wavelength λ₁ over an interrogation area adapted to receive a        biological sample,    -   an optical sensor sensible to a second wavelength λ₂ adapted to        measure the optical radiation at λ₂,    -   a control unit connected to the optical sensor and to the source        of optical radiation wherein said control unit is adapted to        carry out the method of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Nucleosomes are arranged in discrete nanodomains in interphasenuclei of human somatic cells. (A) Super-resolution images of H2B inhuman fibroblast nucleus (hFb, left) and human fibroblast nucleus aftertreatment with trichostatin A (TSA-hFb, right). Progressively higherzooms of the regions inside the red squares are shown next to eachnucleus. (B) Density images showing regions of high (red) and low (blue)H2B density (number of H2B localizations per unit area) in hFb (upper)and TSA-hFb (lower) according to the color scale bar. Afterthresholding, the density images are converted into binary images inwhich regions containing H2B localizations appear white. Every whiteregion is analyzed using a cluster identification algorithm that groupsthe individual localizations based on their proximity into nanodomains.Shown are example nanodomains in hFb (upper) and TSA-hFb (lower) forwhich localizations (crosses) having the same colour belong to the samenanodomain. The centroid position of each nanodomain is shown as a blackdot. The nearest neighbor distances (nnds) between nanodomains insidethe white regions are calculated (double head black arrows), along withthe number of localizations per nanodomain and the nanodomain area. (C)Distribution of the number of H2B localizations per nanodomain,nanodomain area and nnds between nanodomains in hFb (blue) and TSA-hFb(red). Statistical significance between the different distributions isshown as *** (p<10⁻³). See also FIG. 7.

FIG. 2: Nucleosomes are arranged in discrete nanodomains in interphasenuclei of mouse embryonic stem cells. (A-F) Super-resolution images ofH2B in (A) the less pluripotent (Type 1) group of mouse embryonic stemcells (mESCs) cultured in serum plus Lif (sLif) medium, (B) the morepluripotent (Type 2) group of mESCs cultured in sLif medium, (C) mESCscultured in 2iLif medium, (D) neuronal precursor cells (mNPC) obtainedafter differentiation of mESCs, (E) mutant mESCs lacking Tcf3(mESC^(Tcf3−/−)) and (F) mutant mESCs that are triple H1 knockout(mESC^(H1tKO)). Next to each cell type, higher zooms of the regionsinside the red squares are shown. Yellow arrowheads point to brightnanodomains comprising a large number of localizations whereas cyanarrowheads point to dimmer nanodomains comprising a small number oflocalizations. (G-H) Density image showing the differences in nanodomainorganization of mESCs cultured in 2iLif medium (G) and mNPCs (H) Regionsof high (red) and low (blue) H2B density are shown according to thecolor scale bar. (I) Distribution of the number of H2B localizations pernanodomain and nanodomain nnds in mNPCs (blue) and mESCs cultured in2iLif medium (red). Statistical significance is shown as *** (p<10⁻³).See also FIG. 8.

FIG. 3: The number of nucleosomes inside clutches correlates withcellular phenotype. Box plots showing the median number of H2Blocalizations per clutch in hFbs, TSA-hFbs (A); and in different mESCsand in mNPCs (B). (C) Calibration curve to convert from the mediannumber of H2B localizations to the median number of nucleosomes perclutch. The median number of localizations per mononucleosome (redcircle), 12-(green circle) and 24-nucleosome array (black circle)assembled in vitro were used to generate the calibration curve. Purplecircle is data from a 4500 base pair (bp) plasmid assembled intonucleosome-arrays with an expected number of ˜20 nucleosomes per array.Blue circle is data from fluorophore-labeled secondary antibody alone.The median number of localizations per 4500 bp plasmid and per secondaryantibody was interpolated from the calibration curve to obtain themedian number of nucleosomes. Inset shows the first part of the curvecontaining the secondary antibody and the mononucleosomes. Error barscorrespond to standard deviations. (D) Box plots showing the mediannumber of nucleosomes per clutch in hFbs, TSA-hFbs, in the differenttypes of mESCs and in mNPCs. The dotted line corresponds to onenucleosome and the dashed line at 5 nucleosomes separates the morepluripotent cell types from those that are less pluripotent. (E-F) Boxplots showing the median density of nucleosomes per clutch in hFbs,TSA-hFbs (E) in the different types of mESCs and mNPCs (F). For (A, B,D-F) each black dot shows the median number of nucleosomes obtained perindividual nucleus. The red line is the median for the entire populationof nuclei analyzed for that cell type. The light magenta regioncorresponds to the standard error and the dark magenta region to thestandard deviation. Statistical significance between the different celltypes was determined using one-way Anova. The stars indicate p-valuesaccording to * (p<0.05), ** (p<0.01) and *** (p<0.001). See also FIG. 9.

FIG. 4: Clutch size is predictive of pluripotency grade in human inducedpluripotent stem cells clones. (A) Box plots showing the median numberof H2B localizations per clutch in different human induced pluripotentstem cell clones (hiPSCs). (B) Box plots showing the median number ofnucleosomes per clutch in the different hiPSCs. The dotted linecorresponds to one nucleosome. (C) Box plots showing the median densityof nucleosomes per clutch in the different hiPSCs. (D) Pluripotencyscore of the different hiPSCs obtained from the gene card plottedagainst the median number of nucleosomes. Error bars indicate standarddeviations. For black dots, lines, box plot colors and statistics in(A-C) see description in the legend of FIG. 3. See also FIG. 10.

FIG. 5: The linker histone H1 increases in large clutches while RNAPolymerase II associates with small clutches. (A-B) Multi-colorsuper-resolution images showing H2B (red) and H1 (green) in hFb (A) andTSA-hFb (B). Higher zooms of the regions inside white rectangles areshown next to each nucleus. (C) Plot showing the number of H2B (x-axis)and H1 (y-axis) localizations inside clutches for which these twohistones showed colocalization. Error bars in x-axis indicate standarddeviations and in y-axis indicate standard errors. (D) Multi-colorsuper-resolution image showing H2B (red) and RNA polymerase II (PolII11)(green) in TSA-hFb. Progressive zooms of the regions inside whiterectangles are shown below the image of the nucleus. (E) (upper) Plotshowing the distribution of nearest neighbour distances between H2B andPolII11 in hFb (blue) and TSA-hFb (red). (lower) Plot showing the numberof H2B localizations within clutches (y-axis) as a function of thenearest neighbor distances between PolII11 and H2B (x-axis) for hFb(blue) and TSA-hFb (red). Error bars indicate standard errors.

FIG. 6: Computer simulations of nucleosome occupancy. (A) Nucleosomes(light blue) are initially arranged at regular intervals of 50 bp on theDNA fiber (full occupancy). 146 bp of DNA wraps around each nucleosome.(B) A three-dimensional DNA fiber arrangement is generated bypositioning nucleosomes according to a Gaussian chain model withend-to-end distances (le-e) calculated according to the worm like chainmodel (WLM) for a polymer with a persistence length of 150 bp (C) Theresulting DNA fiber configuration is projected onto 2D space (D) In thenucleosome removal (NR) model nucleosomes are removed from the DNA witha given probability ranging from 0 to 95%. When a nucleosome is removed,the linker-DNA length between the neighboring nucleosomes increases by146 bp. (E) In the Linker Length (LL) model the linker-DNA lengths (li)between subsequent nucleosomes are drawn from normal distributions whoseaverages are varied from 50 bp to 3000 bp. (F) Examples of syntheticsuper-resolution images obtained from the simulated arrangement ofnucleosomes at 75%, 57% and 45% nucleosome occupancy. (G) Comparison ofsimulation results for the NR-(black squares and solid line) andLL-Models (white circles and dotted line) to experimental data for hFbs(horizontal blue line) and TSA-hFbs (horizontal red line) at differentlevels of nucleosome occupancy (x-axis). The comparison is made for thenumber of localizations per clutch (upper) nearest neighbor distances(nnds) of clutches (middle) and clutch area (lower). The vertical bluelines and black arrows show the nucleosome occupancy values for whichthe simulation results of the different models intersect theexperimental data for the hFb. Similarly, the vertical red lines andblack arrows show the nucleosome occupancy values for which thesimulation results intersect the experimental data for the TSA-hFb. Seealso FIG. 12.

FIG. 7: Super-resolution imaging of different core histone proteinsunder various fixation and labeling conditions and H3 acetylation.Related to FIG. 1. (A) Comparison of super-resolution (STORM) image ofH2B (upper portion) to conventional fluorescence microscopy image of H2Blabeled with a primary anti-H2B antibody and Alexa Fluor 647-conjugatedsecondary antibody (lower portion, grey). The conventional fluorescenceimage was recorded with 647 nm laser at low enough laser power such thatthe Alexa Fluor 647 fluorophore did not switch off to the dark state.Higher zooms of the region inside the red square are shown to the right.(B) Super-resolution image of core histone protein H3. Higher zoom ofthe region inside the red square is shown as an inset. (C-D)Super-resolution images of H2B after fixation with 4% PFA (C), orMethanol-Ethanol (MeOH-EtOH) (D). Higher zooms of the regions inside thered squares are shown as an inset. (E) Super-resolution image of H2B incells stably expressing H2B fused to SNAP-tag (H2B-SNAP). H2B wasindirectly labeled using an anti-SNAP tag antibody. Higher zoom of theregion inside the red square is shown as an inset. (F-M) Controlexperiment showing the labeling efficiency of H2B antibody in comparisonto the GFP-nanobody. Conventional fluorescence images (grey) of GFP innuclei transiently expressing H2B-GFP are shown for cases in whichGFP-expression was low (F and J) and cases in which GFP-expression washigh (H and L). These nuclei were labeled with either an antibodyagainst H2B (G and I) or a nanobody against GFP (K and M). Correspondingsuper-resolution images obtained from the GFP-nanobody and H2B antibodylabeling are shown next to each nucleus together with higher zooms ofthe regions inside the red rectangles. (N) H3 acetylation in humanfibroblast cells before (hFb, left) and after treatment withtrichostatin A (TSA-hFb, right). Higher zooms of the regions inside thered squares are shown as insets. (O) STORM density image showing all thelocalizations detected (left) and the localizations that are filteredout due to threshold (right). Low (blue) and high (red) density regionsare shown according to the color scale bar (bottom).

FIG. 8: Super-resolution imaging of H2B in mouse embryonic stem cells(mESCs) cultured in serum and Lif (sLif) expressing different levels ofNanog, and H3 acetylation in mESCs. Related to FIG. 2. (A) Conventionalfluorescence (Cony Fl) image of Nanog (grey) labeled with a primaryanti-Nanog antibody and Cy3-conjugated secondary antibody. Densityimages show high (red) and low (blue) H2B density regions in H2Bsuper-resolution images of Type 1 (upper) and Type 2 (lower) mESCscultured in sLif according to the color scale bar. (B-F)Super-resolution images of H3 acetylation in mESC^(Tcf3−/−) (B), mESCscultured in sLif, Type 1 (C), mESCs cultured in 2iLif, (D), mESCscultured in sLif, Type 2 (E), and mouse neuronal precursor cells (mNPC)(F).

FIG. 9: In vitro experiments for the calibration curve. Related to FIG.3. (A) Super-resolution images of fluorophore-labeled secondaryantibodies (2aryAb), mononucleosomes, 12- and 24-nucleosome arrays (leftand middle) and examples of identified nanodomains in these imagesrepresented as colored crosses (right). (B) Plot showing the cumulativedistribution of the number of localizations per fluorophore-labeledsecondary antibody (blue line), mononucleosome (red line), 12-(greenline) and 24-nucleosome array (black line). The median numbers oflocalizations from these distributions (dashed lines) were used in thecalibration curve to convert from the number of H2B localizations to thenumber of nucleosomes. (C) Example electron microscopy images obtainedfrom the assembled 12-nucleosome array.

FIG. 10: Classical characterization of the human induced pluripotentstem cell (hiPSC) clone 8 and 13. Related to FIG. 4. (A-B) hiPSCs #8 and#13 were visualized by phase contrast, were stained for the expressionof the stem cell marker alkaline phosphatase (AP) (A) and werevisualized by conventional fluorescence microscopy after staining withantibodies against the stem cell markers TRA 1-60, SSEA4 (A), Oct4, Sox2and Nanog (B). Conventional fluorescence microscopy using an anti-Oct4antibody was carried out in single cells. Mean intensity of Oct4 signalin hiPSCs #8 is 144±1 (Mean intensity±SEM) and in #13 is 2063±7 (Meanintensity±SEM). Nuclei were stained with DAPI. (C) Embryoid bodies(upper panels) were formed from hiPSCs #8 and #13 and induced todifferentiate in the three germ layers. Immunostaining using anti-Foxa2(endoderm), anti-α-Sma (mesoderm) and anti-β-Tubulin III (ectoderm) werecarried out 15 days after differentiation. (D) 7-weeks teratomas formedfrom hiPSCs #8 and #13 were weighted, sectioned and stained withhematoxylin and eosin to identify tissues derived from the threedifferent germ layers. (E) Box plot showing the pluripotency scoreaccording to the gene card technology of all the hiPSC clones (#8 green,#16 pink, #20 brown, #6 cyan and #13 yellow) analyzed. Box indicateslower (25%) and upper (75%) percentile range, the line represents themedian pluripotency score and the whiskers represent minimum and maximumpluriotency scores resulting from the quantification of 67 differentcells.

FIG. 11: Quantification of the number of H2B and H1 localizations inmESCs. Related to FIG. 5. (A) Plot showing the number of H2B (x-axis)and H1 (y-axis) localizations inside clutches for which these twohistones showed colocalization in mESCs cultured in sLif (blue) andmESCs^(H1tKO) (red). (B) Bar plot showing the average number of H1localizations detected per area in the nuclei of mESCs cultured in sLif(blue) and mESCs^(H1tKO) (red). (A-B) Error bars in x-axis indicatestandard deviations in y-axis indicate standard errors.

FIG. 12: Computer simulations of H2B labeling efficiency. Related toFIG. 6. (A) Example synthetic super-resolution images obtained fromcomputer simulations of 20%, 50% and 80% H2B labeling efficiency. 100%labeling efficiency corresponds to an average of 1.6 antibodies pernucleosome as determined from in vitro images of fluorophore-labeledsecondary antibodies and mononucleosomes. (B) Comparison of simulationresults (black circles and black line) to experimental data for hFb(horizontal blue line) and hFb-TSA (horizontal red line) at differentlevels of antibody labeling efficiency (x-axis). The comparison is madefor the number of localizations per clutch (upper), nearest neighbordistances of clutches (middle) and clutch area (lower). The verticalblue lines show the antibody labeling efficiency values for which thesimulation results intersect the experimental data for the hFb.Similarly, the vertical red lines show the antibody labeling efficiencyvalues for which the simulation results intersect the experimental datafor the TSA-hFb. (C-E) Comparison of the distributions for the number oflocalizations per clutch (C), clutch nearest neighbor distances (D) andclutch areas (E) between experimental data and occupancy simulations(57% occupancy for hFb and 45% occupancy for TSA-hFb). The distributionsobtained from the experimental data are shown as blue lines for hFb andred lines for TSA-hFb in all cases. The distributions obtained from thesimulations are shown as the cyan lines for hFb and magenta lines forTSA-hFb. Statistical comparisons of experimental and simulateddistributions provided a r2 of 0.95 and 0.95 (number of localizations,hFb and TSA-hFb respectively), 0.97 and 0.98 (clutch nearest neighbordistances, hFb and TSA-hFb respectively) and 0.92 and 0.97 (clutchareas, hFb and TSA-hFb, respectively).

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have resolved how nucleosomes arearranged along the chromatin fiber in a large number of different celltypes. Their observations indicate that nucleosomes are grouped indiscrete domains, which they termed “nucleosome clutches” in analogywith “egg clutches” (Example 1). They developed quantitative methods toassess clutch size, defined as the number of nucleosomes per clutch, andfound that this number is very heterogeneous in a given nucleus arguingagainst the existence of a well-organized and ordered fiber. Bycomparing experimental data to computer simulations they estimated thenucleosome occupancy of the chromatin fiber and found thatnucleosome-depleted regions intersperse nucleosome clutches. Two-colorsuper-resolution imaging showed increased levels of H1 in largerclutches containing more nucleosomes suggesting that H1 might beresponsible for bringing nucleosomes into close proximity inside theclutches. On the other hand, RNA Polymerase II associated more closelywith smaller clutches containing fewer nucleosomes, suggesting that thechromatin fiber within these regions is more accessible (Example 5).Strikingly, despite the heterogeneity in the clutch size in a givennucleus, on average differentiated cells contained clutches with largernumber of nucleosomes compared to stem cells. Furthermore, there was ahigh degree of correlation between clutch size and pluripotency grade ofwild-type and mutant mouse embryonic stem cells (mESC) cultured underdifferent conditions and pluripotency grade of a number of differenthuman induced pluripotent stem cell (hiPSC) clones. Therefore,nucleosome organization is predictive of cell pluripotency (Example 4).These results open up exciting possibilities for identifying stem cellsby analyzing their nucleosome arrangement organization is predictive ofcell pluripotency. Thus, the inventors have developed a method foridentifying the chromatin state of a cell by analyzing their nucleosomearrangement.

Method for Detecting the Chromatin State of a Cell

According to the “textbook picture”, chromatin compaction follows ahierarchical model where nucleosomes from a “beads-on-string” fiber of10 nm in diameter, which folds into higher ordered fibers of 30 nm,which in turn compact progressively into larger fibers of 100-200 nm.

The structural information needs to be obtained using optical meanshaving optical sensors combined with post-processing software configuredto reveal internal structures having a length scale of about 10 nm.

In a first aspect, the invention relates to a method for detecting thechromatin state of a cell comprising,

-   -   a) contacting a sample containing cells with a first antibody        capable of specifically binding to a histone protein,    -   b) contacting the antibody:histone complex formed in step a)        with a secondary antibody having at least one photoswitchable        fluorophore adapted to be optically excited at a certain        wavelength λ₁ and to emit light at a wavelength λ₂ different        from λ₁,    -   c) recording a super resolution image of nucleosome organization        by means of a sensor being sensitive at least to the wavelength        of emission of the photoswitchable fluorophore by exciting the        sample with an optical radiation having a wavelength λ₁,    -   d) correlating the image obtained in step c) with size of        nucleosomal clutches, nucleosomal density and/or number of        nucleosomes per nucleosomal clutches, and    -   e) comparing data obtained in step d) with a corresponding        reference value to obtain a score based on size of nucleosomal        clutches, nucleosomal density and/or number of nucleosomes per        nucleosomal clutch,

wherein if the cell comprises smaller clutches, less densely compactednucleosomes or less nucleosomes per clutches compared to thecorresponding reference value is indicative that said cell is in an openchromatin state and wherein if the cell comprises bigger clutches, moredensely compacted nucleosomes or more nucleosomes per clutches comparedto the corresponding reference value is indicative that said cell is ina close chromatin state.

Detecting, as used herein, refers both to determine and/or identify if acell is in an open or close chromatin state. As will be understood bythose skilled in the art, the detection, although preferred to be, neednot be correct for 100% of the cells to be detected or evaluated. Theterm, however, requires that a statistically significant portion ofcells can be identified as in an open chromatin state or in a closechromatin state. Whether a cell is statistically significant can bedetermined without further ado by the person skilled in the art usingvarious well known statistic evaluation tools, e.g., determination ofconfidence intervals, p-value determination, Student's t-test,Mann-Whitney test, etc. Details are found in Dowdy and Wearden,Statistics for Research, John Wiley & Sons, New York 1983. Preferredconfidence intervals are at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%. The p-values are, preferably,0.05, 0.01, 0.005 or lower.

As the skill person can understand, the method of the invention allowscomparing the chromatin state between two cells, and thus it is possibleto determine if two cells have a similar or different chromatin state.

“Chromatin state of a cell”, as used herein relates to a condition of acell showing open (active) chromatin or close (inactive) chromatin. Saidterms are known by a skill person. “Open chromatin” means a DNA in whichhistone modifications such as acetylation lead to exposure of a DNAsequence thus allowing binding of transcription factors andtranscription to take place. Open chromatin is structurally loose toallow access to RNA and DNA polymerases that transcribe and replicatethe DNA. “Close chromatin” is found associated with structural proteinsand include modifications of the histone tails that lead to are moretightly packaged state of the chromatin, which is less accessible to thebinding of the majority of transcription factors and polymerases.

In a preferred embodiment the cell in an open chromatin state isselected from the group consisting of transcriptionally active cells,pluripotent cells, cancer cells and drug perturbed cells. In a morepreferred embodiment, the cell in an open chromatin state is apluripotent cell. The term “transcriptionally active cell” as usedherein, relates to a cell having an active chromatin, which means a DNAin which histone modifications such as acetylation lead to exposure of aDNA sequence thus allowing binding of transcription factors andtranscription to take place.

“Pluripotent cell” as used herein, relates to a primordial cell that candifferentiate into a sub-group of specialized types of cells, forexample, a stem cell that has the potential to differentiate into any ofthe three germ layers: endoderm (interior stomach lining,gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood,urogenital), or ectoderm (epidermal tissues and nervous system).Pluripotent stem cells can give rise to any fetal or adult cell type.However, alone they cannot develop into a fetal or adult animal becausethey lack the potential to contribute to extraembryonic tissue, such asthe placenta. Illustrative, non-limitative examples of pluripotent cellsinclude adipose-derived stem cells (ASCs), amniotic stem cells, bonemarrow-derived stem cells (BMSCs), cord blood-derived stem cells(CBSCs), embryonic stem cells (ESCs), fetal stem cells (FSCs), amnioticstem cells, endothelial stem cells, epidermal stem cells, haematopoieticstem and progenitor cells (HSPCs), mesenchymal stem cells (MSCs), neuralstem cells (NSCx), retinal stem and progenitor cells (RSPCs), etc.

In a further embodiment, the pluripotent cell is an induced pluripotentstem cell, commonly abbreviated as iPS cell or iPSC, which is a type ofpluripotent stem cell artificially derived from a non-pluripotent cell,typically an adult somatic cell, by inducing a “forced” expression ofspecific genes. iPSCs are similar to natural pluripotent stem cells inmany respects, such as the expression of certain stem cell genes andproteins, chromatin methylation patterns, doubling time, embryoid bodyformation, teratoma formation, viable chimera formation, and potency anddifferentiability.

“Cancer cell” refers to a cell from a cancer or tumor or a cancer cellline. “Cancer” refers to a broad group of diseases involving unregulatedcell growth and which are also referred to as malignant neoplasms. Theterm is usually applied to a disease characterized by uncontrolled celldivision (or by an increase of survival or apoptosis resistance) and bythe ability of said cells to invade other neighboring tissues (invasion)and spread to other areas of the body where the cells are not normallylocated (metastasis) through the lymphatic and blood vessels, circulatethrough the bloodstream, and then invade normal tissues elsewhere in thebody. Depending on whether or not they can spread by invasion andmetastasis, tumours are classified as being either benign or malignant:benign tumours are tumours that cannot spread by invasion or metastasis,i.e., they only grow locally; whereas malignant tumours are tumours thatare capable of spreading by invasion and metastasis. Biologicalprocesses known to be related to cancer include angiogenesis, immunecell infiltration, cell migration and metastasis. Cancers usually sharesome of the following characteristics: sustaining proliferativesignalling, evading growth suppressors, resisting cell death, enablingreplicative immortality, inducing angiogenesis, and activating invasionand eventually metastasis. Cancers invade nearby parts of the body andmay also spread to more distant parts of the body through the lymphaticsystem or bloodstream. Cancers are classified by the type of cell thatthe tumour cells resemble, which is therefore presumed to be the originof the tumour. These types include:

-   -   Carcinoma: Cancers derived from epithelial cells. This group        includes many of the most common cancers, particularly in the        aged, and include nearly all those developing in the breast,        prostate, lung, pancreas, and colon.    -   Sarcoma: Cancers arising from connective tissue (i.e. bone,        cartilage, fat, nerve), each of which develop from cells        originating in mesenchymal cells outside the bone marrow.    -   Lymphoma and leukaemia: These two classes of cancer arise from        hematopoietic (blood-forming) cells that leave the marrow and        tend to mature in the lymph nodes and blood, respectively.    -   Germ cell tumour: Cancers derived from pluripotent cells, most        often presenting in the testicle or the ovary (seminoma and        dysgerminoma, respectively).    -   Blastoma: Cancers derived from immature “precursor” cells or        embryonic tissue. Blastomas are more common in children than in        older adults.

In a preferred embodiment the cancer cells are cells from a cancerselected from breast, ovarian, prostate, brain, pancreas, skin, bone,bone marrow, blood, thymus, uterus, testicles, hepatobiliary and livertumors, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma,germinoma, glioblastoma, glioma, hemangioendothelioma, hemangio sarcoma,hematoma, hepatoblastoma, leukaemia, lymphoma, medulloblastoma,melanoma, neuroblastoma, hepatobiliary cancer, osteosarcoma,retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma,acrallentiginous melanoma, actinic keratosis adenocarcinoma, adenoidcystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma,astrocytictumors, bartholin gland carcinoma, basal cell carcinoma,bronchial gland carcinoma, capillary carcinoid, carcinoma,carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor,endometrial hyperplasia, endometrial stromal sarcoma, endometrioidadenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodularhyperplasia, germ cell tumors, glioblastoma, glucagonoma,hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma,hepatic adenomatosis, hepatocellular carcinoma, hepatobiliary cancer,insulinoma, intraepithelial neoplasia, interepithelial squamous cellneoplasia, invasive squamous cell carcinoma, large cell carcinoma,leiomyosarcoma, melanoma, malignant melanoma, malignantmesothelialtumor, medulloblastoma, medulloepithelioma, mucoepidermoidcarcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodularmelanoma, osteosarcoma, papillary serous adenocarcinoma, pituitarytumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cellcarcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma,small cell carcinoma, soft tissue carcinoma, somatostatin-secretingtumor, squamous carcinoma, squamous cell carcinoma, undifferentiatedcarcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilm's tumor,intracerebral cancer, head and neck cancer, rectal cancer, astrocytoma,glioblastoma, small cell cancer, and non-small cell cancer.

“Drug perturbed cell”, as used herein relates to a cell treated with acompound that target the cell machinery of transcription, the cell cycleor proliferation process. Illustrative non-limitative examples ofcomponents of the transcription machinery are RNA polymerase;specificity factors (alter the specificity of RNA polymerase for a givenpromoter or set of promoters, making it more or less likely to bind tothem (i.e. sigma factors used in prokaryotic transcription); repressors(bind to non-coding sequences on the DNA strand that are close to oroverlapping the promoter region, impeding RNA polymerase's progressalong the strand, thus impeding the expression of the gene; generaltranscription factors (position RNA polymerase at the start of aprotein-coding sequence and then release the polymerase to transcribethe mRNA); activators (enhance the interaction between RNA polymeraseand a particular promoter, encouraging the expression of the gene.Activators do this by increasing the attraction of RNA polymerase forthe promoter, through interactions with subunits of the RNA polymeraseor indirectly by changing the structure of the DNA); enhancers (sites onthe DNA helix that are bound to by activators in order to loop the DNAbringing a specific promoter to the initiation complex); silencers(regions of DNA that are bound by transcription factors in order tosilence gene expression); chromatin remodeling through specific use ofmiRNA molecules presents one method by which euchromatin, typicallyassociated with transcriptional activity, is converted toheterochromatin, reducing transcription. This occurs by means of RNAinduced transcriptional silencing complex or “RITS.”

Illustrative non-limitative examples of such drugs are tamoxifene,bicalutamide and various types of anti-inflammatory and anabolicsteroid, enzyme inhibitors such as kinase and acetylase inhibitors oractivators. As a result of treatment with the drug the cell could suffertranscription of a gene.

Step a) of the method for detecting the chromatin state of a cellcomprises contacting a sample containing cells with a first antibodycapable of specifically binding to a histone protein. Thus, according toan embodiment of the invention, an antibody:histone complex is formedcontacting a sample containing cells with a first antibody capable ofspecifically binding to a histone protein.

“Sample”, as used herein refers to any biological sample susceptible ofcontaining cells, and it can be obtained by conventional methods knownby those of average skill in the art, depending on the nature of thesample.

In a particular embodiment, said biological sample is a biopsy sample,tissue, cell or biofluid sample (plasma, serum, saliva, semen, sputum,cerebral spinal fluid (CSF), tears, mucus, sweat, milk, brain extractsand the like). Said biological samples can be obtained by anyconventional method. In another aspect, the sample is a cell culturesample.

In a more preferred embodiment, the sample is a mouse or humancommercial cell line. In another preferred embodiment, the sample is abiopsy sample from a human patient. In another preferred embodiment, thesample comprises primary cells purified from body parts of human donors.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules containing an antigen fixing site binding specifically(immunoreacting) with an antigen, such as a protein for example. Thereare 5 isotypes or main classes of immunoglobulins: immunoglobulin M(lgM), immunoglobulin D (lgD), immunoglobulin G (lgG), immunoglobulin A(lgA) and immunoglobulin E (lgE).

The antibodies that are going to be used in the present invention canbe, for example, polyclonal sera, hybridoma supernatants or monoclonalantibodies, antibody fragments, Fv, Fab, Fab′ and F(ab′)2, scFv,diabodies, triabodies, tetrabodies and humanized antibodies.

The suitable conditions for the formation of the antibody:histonecomplex to take place are known by the skilled in the art. If the samplecontaining cells contains histone proteins, then the correspondingantibody:histone complex will be formed.

“Histone protein”, as used herein relates to a highly alkaline proteinfound in eukaryotic cell nuclei that packages and orders the DNA intostructural units called nucleosomes. Five major families of histonesexist: H1/H5, H2A, H2B, H3 and H4. Histones H2A, H2B, H3 and H4 areknown as the core histones, while histones H1 and H5 are known as thelinker histones. “Nucleosomes” are a repeating unit of the chromatin,formed by 146 base pairs (bp) of DNA wrapped around octamers of the fourcore histone proteins (H2A, H2B, H3 and H4).

As a person skilled in the art can know, the histone protein can also bedetected by detecting a functionally equivalent variant of a histoneprotein.

“Functionally equivalent variant” is understood to mean all thoseproteins derived from a histone sequence by modification, insertionand/or deletion or one or more amino acids, whenever the function issubstantially maintained.

Assays to determine the function of an enzyme are known by the skilledperson and include, without limitation, initial rate assays, progresscurve assays, transient kinetics assays and relaxation assays.Continuous assays of enzymatic activity include, without limitation,spectrophotometric, fluorometric, calorimetric, chemiluminiscent, lightscattering and microscale thermopheresis assays. Discontinuous assays ofenzymatic activity include, without limitation, radiometric andchromatographic assays. As the skilled person understands, factors thatmay influence enzymatic activity comprise salt concentration,temperature, pH, and substrate concentration.

The function of a histone can be determined by analyzing the compactionof DNA. The compaction of DNA can be assay using several methods knownin the art, by way of illustrative-non limitative example by densitygradient centrifugation on MNase digested samples, comet assay.Particularly the function of H2B can be assayed by determining thephosphorylation of H2B at serine 14, which is linked to chromatincondensation. Additionally, by way of illustrative-non limitativeexample, the function of the H2B can be assayed by detecting acetylationin Lys12 and in Lys15 or ubiquitylation in Lys120, all of thesemodifications, associated with transcriptionally activation, and thuswith an open chromatin state.

Preferably, variants of a histone protein are (i) polypeptides in whichone or more amino acid residues are substituted by a preserved ornon-preserved amino acid residue (preferably a preserved amino acidresidue) and such substituted amino acid may be coded or not by thegenetic code, (ii) polypeptides in which there is one or more modifiedamino acid residues, for example, residues modified by substituentbonding, (iii) polypeptides resulting from alternative processing of asimilar mRNA, (iv) polypeptide fragments and/or (v) polypeptidesresulting from a histone fusion or the polypeptide defined in (i) to(iii) with another polypeptide, such as a secretory leader sequence or asequence being used for purification (for example, His tag) or fordetection (for example, Sv5 epitope tag). The fragments includepolypeptides generated through proteolytic cut (including multisiteproteolysis) of an original sequence. The variants may bepost-translationally or chemically modified. Such variants are supposedto be apparent to those skilled in the art.

As known in the art, the “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and the substitutedamino acids preserved from a polypeptide with the sequence of a secondpolypeptide. The variants are defined to include polypeptide sequencesdifferent from the original sequence, preferably different from theoriginal sequence in less than 40% of residues per segment concerned,more preferably different from the original sequence in less than 25% ofresidues per segment concerned, more preferably different from theoriginal sequence in less than 10% of residues per segment concerned,more preferably different from the original sequence in only a fewresidues per segment concerned and, at the same time, sufficientlyhomologous to the original sequence to preserve functionality of theoriginal sequence. The present invention includes amino acid sequenceswhich are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95%similar or identical to the original amino acid sequence. The degree ofidentity between two polypeptides may be determined using computeralgorithms and methods which are widely known to those skilled in theart. The identity between two amino acid sequences is preferentiallydetermined using BLASTP algorithm [BLASTManual, Altschul, S. et al.,NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990)].

In a preferred embodiment, the histone protein is core histone protein.

“Core histone protein”, as used herein, refers to any histone selectedfrom the group consisting of histone H2A, H2B, H3 and H4. In a morepreferred embodiment, the core histone protein is H2B.

“H2B”, as used herein, refers to one of the 5 main histone proteinsinvolved in the structure of chromatin in eukaryotic cells. Featuring amain globular domain and a long N terminal tail H2B is involved with thestructure of the nucleosomes of the ‘beads on a string’ structure. H2Bhas 19 variants in humans. The detection of any variant of H2B can beused in the present invention.

As the person skilled in the art understands it may be necessary that,after contacting the sample with the first antibody, the sample isproperly collected, fixed and/or sectioned. Cells in a sample can befixed by any suitable process including perfusion or by submersion in afixative. Fixatives can be classified as cross-linking agents (such asaldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, aswell as non-aldehyde cross-linking agents), oxidizing agents (e.g.,metallic ions and complexes, such as osmium tetroxide and chromic acid),protein-denaturing agents (e.g., acetic acid, methanol, and ethanol),fixatives of unknown mechanism (e.g., mercuric chloride, acetone, andpicric acid), combination reagents (e.g., Carnoy's fixative, methacarn,Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid),microwaves, and miscellaneous fixatives (e.g., excluded volume fixationand vapor fixation). Additives may also be included in the fixative,such as buffers, detergents, tannic acid, phenol, metal salts (such aszinc chloride, zinc sulfate, and lithium salts), and lanthanum. In apreferred embodiment, the fixative used in the present invention is acombination of methanol and ethanol, more particularly in a 1:1 ratio.

To reduce background staining, samples can be incubated with a bufferthat blocks the reactive sites to which the primary or secondaryantibodies may otherwise bind. Common blocking buffers include normalserum, non-fat dry milk, BSA, or gelatin. Commercial blocking bufferswith proprietary formulations are available. Methods to eliminatebackground staining include dilution of the primary or secondaryantibodies, changing the time or temperature of incubation, or using adifferent primary antibody. In a preferred embodiment, the blocking iscarry out by a buffer comprising BSA

The detection of the antibody:histone complex (step b) is carried out bycontacting said complex with a secondary antibody, having at least onephotoswitchable fluorophore adapted to be optically excited at a certainwavelength λ₁ and to emit light at a wavelength λ₂ different from λ₁.When the sample having the antibody:histone complex is excited withoptical energy, for instance by means of a laser beam of a wavelengthλ₁, those locations of the antibody:histone complex linked to thephotoswitchable fluorophore emit light at the wavelength λ₂.

“Fluorophore”, as used herein, refers to entities that can emit light ofa certain emission wavelength when exposed to a stimulus, for example,an excitation wavelength.

“Photoswitchable” as used herein, relates to an entity which can beswitched between different light-emitting or non-emitting states byincident light of different wavelengths. Typically, a “switchable”entity can be identified by one of ordinary skill in the art bydetermining conditions under which an entity in a first state can emitlight when exposed to an excitation wavelength, switching the entityfrom the first state to the second state, e.g., upon exposure to lightof a switching wavelength, then showing that the entity, while in thesecond state can no longer emit light (or emits light at a reducedintensity) or emits light at a different wavelength when exposed to theexcitation wavelength. Examples of switchable entities are disclosed inWO 2008/091296. As a non-limiting example of a switchable fluorophore,Cy5 can be switched between a fluorescent and a dark state in acontrolled and reversible manner by light of different wavelengths,e.g., 633 nm or 657 nm red light can switch or deactivate Cy5 to astable dark state, while 405 nm or 532 nm light can switch or activatethe Cy5 back to the fluorescent state.

In some cases, the fluorophore can be reversibly switched between thetwo or more states, e.g., upon exposure to the proper stimuli. Forexample, a first stimuli (e.g., a first wavelength of light) may be usedto activate the switchable fluorophore, while a second stimuli (e.g., asecond wavelength of light) may be used to deactivate the switchablefluorophore, for instance, to a non-emitting state. Any suitable methodmay be used to activate the fluorophore. For example, in one embodiment,incident light of a suitable wavelength may be used to activate theentity to emit light, i.e., the entity is photoswitchable. Thus, thephotoswitchable fluorophore can be switched between differentlight-emitting or non-emitting states by incident light, e.g., ofdifferent wavelengths. The light may be monochromatic (e.g., producedusing a laser) or polychromatic.

In another embodiment, the entity may be activated upon stimulation byelectric field and/or magnetic field. In other embodiments, the entitymay be activated upon exposure to a suitable chemical environment, e.g.,by adjusting the pH, or inducing a reversible chemical reactioninvolving the entity, etc.

Similarly, any suitable method may be used to deactivate the entity, andthe methods of activating and deactivating the entity need not be thesame. For instance, the entity may be deactivated upon exposure toincident light of a suitable wavelength, or the entity may bedeactivated by waiting a sufficient time.

In some embodiments, the switchable entity includes a first,light-emitting portion (e.g., a fluorophore), and a second portion thatactivates or “switches” the first portion.

Upon exposure to light, the second fluorophore may activate the firstfluorophore a, causing the first fluorophore to emit light. Examples ofactivator fluorophores include, but are not limited to Alexa Fluor 405(Invitrogen), Alexa 488 (Invitrogen), Cy2 (GE Healthcare), Cy3 (GEHealthcare), Cy3.5 (GE Healthcare), or Cy5 (GE Healthcare), or othersuitable dyes. Examples of light-emitting portions include, but are notlimited to, Cy5, Cy5.5 (GE Healthcare), or Cy7 (GE Healthcare), AlexaFluor 647 (Invitrogen), or other suitable dyes. These may linkedtogether, e.g., covalently, for example, directly, or through a linker,e.g., forming compounds such as, but not limited to, Cy5-Alexa Fluor405, Cy5-Alexa Fluor 488, Cy5-Cy2, Cy5-Cy3, Cy5-Cy3.5, Cy5.5-Alexa Fluor405, Cy5.5-Alexa Fluor 488, Cy5.5-Cy2, Cy5.5-Cy3, Cy5.5-Cy3.5, Cy7-AlexaFluor 405, Cy7-Alexa Fluor 488, Cy7-Cy2, Cy7-Cy3, Cy7-Cy3.5, or Cy7-Cy5.In a more preferred embodiment the first fluorophore (activator) isAlexa 405 and the second fluorophore is Alexa 647.

In another preferred embodiment, wavelength λ₁ is 647 nm, wavelength λ₂is 670 nm and wavelength λ₃ is 405 nm.

Any suitable method may be used to link the first, light-emittingfluorophore and the second, activation fluorophore. In some cases, alinker is chosen such that the distance between the first and secondfluorophore is sufficiently close to allow the activator fluorophore toactivate the light-emitting fluorophore as desired, e.g., whenever thelight-emitting fluorophore has been deactivated in some fashion.Typically, the fluorophore will be separated by distances on the orderof 500 nm or less, for example, less than about 300 nm, less than about100 nm, less than about 50 nm, less than about 20 nm, less than about 10nm, less than about 5 nm, less than about 2 nm, less than about 1 nm,etc. Examples of linkers include, but are not limited to, carbon chains(e.g., alkanes or alkenes), polymer units, or the like.

The switchable entity may comprise a first fluorophore directly bondedto the second fluorophore, or the first and second entity may beconnected via a linker or a common entity. Whether a pair of lightemitting portion and activator portion produces a suitable switchableentity can be tested by methods known to those of ordinary skills in theart. For example, light of various wavelength can be used to stimulatethe pair and emission light from the light-emitting portion can bemeasured to determine whether the pair makes a suitable switch.

Additional details about fluorophores can be found in WO2009/085218.

Step c) of the method of the invention comprises recording a superresolution image of nucleosome organization by means of a sensor beingsensitive at least to the wavelength of emission of the photoswitchablefluorophore by exciting the sample with an optical radiation having awavelength λ₁. Recording an image by means of an optical sensor aimingto the optically excited sample provides a bitmap image at certainresolution having information about the nucleosome organization. Inparticular, the image shows the projection over the focal plane of thesensor used for recording the image of the location of thephotoswitchable fluorophores that have emitted light. This informationwill be used to provide characteristic length scales and density of somerelevant structural parts of the protein that allows identifyingnucleosomal organization and thus the chromatin state of a cell.

“Super resolution image” as used herein, refers to an image with anaxial and lateral resolution under 100 nm allowing single moleculelocalization. At present, super resolution images provides a resolutionnear the limit of the length scale defined by chromatin fibers, that is10-30 nm.

In a preferred embodiment, the images obtained are characterized by alateral (XY) resolution of approximately 20-30 nm and axial (Z)resolution of 50-60 nm.

The super resolution images can be obtained by any super resolutiontechniques known in the art. Super-resolution techniques allow thecapture of images with a higher resolution than the diffraction limit.They fall into two broad categories, “true” super-resolution techniques,which capture information contained in evanescent waves, and“functional” super-resolution techniques, which use clever experimentaltechniques and known limitations on the matter being imaged toreconstruct a super-resolution image. There are two major groups ofmethods for functional super-resolution microscopy:

1. Deterministic super-resolution: The most commonly used emitters inbiological microscopy, fluorophores, show a nonlinear response toexcitation, and this nonlinear response can be exploited to enhanceresolution. These methods include without limitation STED, GSD, RESOLFTand SSIM.

2. Stochastical super-resolution: The chemical complexity of manymolecular light sources gives them a complex temporal behaviour, whichcan be used to make several close-by fluorophores emit light at separatetimes and thereby become resolvable in time. These methods includewithout limitation SOFI and all single-molecule localization methods(SMLM) such as SPDM, SPDMphymod, PALM, FPALM, STORM and dSTORM.

In a preferred embodiment, the super resolution image is obtained by astochastical super resolution technique, preferably STORM, PALM and(PALM, and more preferably by STORM. STORM combines two concepts: singlemolecule localization and fluorophore photoswitching. The first conceptallows one to localize the position of a single fluorophore withnanometer precision. Photoswitching makes it possible to “turn off” mostfluorophores into a dark state and “turn on” only a small subset of themat a time. As a result, the images of the “active” fluorophores areisolated in space and their positions can be localized with highprecision. Once all the fluorophores are imaged and their positions arelocalized, a high-resolution image can be reconstructed from theselocalizations. To date, the spatial resolution achieved by thistechnique is ˜20 nm in the lateral dimensions and ˜50 nm in the axialdimension. More details of STORM technology are described inWO2013090360, WO2009085218 and EP2378343.

In a preferred embodiment, a plurality of super resolution images aretaken by means of a sensor being sensitive at least to the wavelength ofemission of the second fluorophore λ₂ rendering a further superresolution image by collecting the sensed light emissions recorded inthe plurality of images. According to said further embodiment, aplurality of images are recorded and post-processed in order to obtain anew image with the accumulated value of the optical radiation emitted bythe sample. When a sample is exited with an optical radiation some ofthe photoswitchable fluorophores are activated and other photoswitchablefluorophores are not. The new image provides information of a largenumber of locations of photoswitchable fluorophores because theprobability of recording the emission of light of certainphotoswitchable fluorophore being excited is higher.

According to an embodiment, a pair of different photoswitchablefluorophores is used. The first photoswitchable fluorophore is adaptedto be optically excited at a certain wavelength λ₁ and to emit light ata wavelength λ₂ different from λ₁; and, the second photoswitchablefluorophore is adapted to be optically excited at a wavelength λ₃ andreactivate the first fluorophore by bringing it from its dark state backto its ground state.

Thus, in a preferred embodiment of the method of the invention, thesecondary antibody further comprises a second fluorophore adapted to beoptically excited at a wavelength λ₃ and reactivate the firstfluorophore by bringing it from its dark state back to its ground state,upon which the first fluorophore can be excited again at its excitationwavelength λ₁ and emit light at its emission wavelength λ₂.

In this case, a first step the sample is excited with an opticalradiation having a wavelength λ₁ turning the first fluorophore to a darkstate. A further optical radiation having a wavelength λ₃ excites thesecond photoswitchable fluorophore which reactivates the firstfluorophore by bringing it from its dark state back to its ground state,upon which the first fluorophore can be excited again at its excitationwavelength λ₁ and emit light at its emission wavelength λ₂. This lastexcitation using an optical radiation at a wavelength λ₁ provides theemission at an emission wavelength λ₂ that is recorded at least in oneimage.

In a preferred embodiment, the power of the optical radiation having awavelength λ₃ is monotonically increased. In an example, the opticalradiation at a wavelength λ₃ has been gradually increased in a sigmoidalmanner reaching a maximum power value, keeping this maximum value untilthe fluorophores are exhaustively imaged and photobleached.

In another preferred embodiment before recording each super resolutionimage of the plurality of super resolution images, the sample is excitedonce or more times with an optical radiation having a wavelength λ₁ andsubsequently excited once or more times with an optical radiation havinga wavelength λ₃.

As the skill person knows, during imaging, only an optically resolvablesubset of fluorophores is activated to a fluorescent state at any givenmoment, such that the position of each fluorophore can be determinedwith high precision by finding the centroid position of thesingle-molecule images of particular fluorophore. The fluorophore issubsequently deactivated, and another subset is activated and imaged.Iteration of this process allows numerous fluorophores to be localizedand a super-resolution image to be constructed from the image data.

One fluorophore is recorded in the image by a plurality of pixelsgrouped in a region of the said image. The value of each pixel isassociated to a certain value of radiation. The location of thefluorophore needs to be determined for the set of pixels havinginformation of that fluorophore.

A more complex situation is found when two or more fluorophores areclose enough as for a plurality of pixels show the accumulated radiationof the plurality of fluorophores. That is, the radiation valuerepresented in a single pixel may be the contribution of the radiationfrom more than one fluorophore.

The individual locations of photoswitchable fluorophores and clusterinformation need to be identified over the image.

Step d) of the present invention, comprises correlating the imageobtained in step c) with size of nucleosomal clutches, nucleosomaldensity and/or number of nucleosomes per nucleosomal clutches.

“Nucleosome clutch”, as used herein relates to a heterogeneousnucleosome group.

“Size of nucleosomal cluches” as used herein relates to the number ofnucleosomes per clutch.

“Nucleosomal density”, as used herein, relates to the number ofnucleosomes in a clutch divided by the unit area of that clutch.

Thus, according to the invention, the image obtained in step c) isconverted to a list of “fluorescent probe positions”. Several knownsoftwares can be used for obtaining fluorescent probe positions, asillustrative non-limiting example the Insight 3 provided by BO Huang,University of California, San Francisco. Briefly, peaks insingle-molecule images are identified based on a threshold and fit to asimple Gaussian to determine the x and y positions. The final images arerendered by representing each x-y position (localization) as a Gaussianwith a width that corresponds to the determined localization precision(9 nm). Sample drift during acquisition is calculated and subtracted byreconstructing STORM images from subsets of frames (typically 500-1000frames, for which drift was assumed to be small) and correlating theseimages to a reference frame (typically one that is reconstructed at theinitial time segment). For multicolor images, each peak is color codedbased on whether the emission is recorded immediately after λ₃ oranother activation wavelength (λ₄). The peaks coming from a frame notbelonging to the one right after an activation frame were coded as“non-specific”. A crosstalk algorithm as described previously is appliedto correct for non-specific activations by the imaging laser (Dani etal., 2010). Briefly, the number of “apparent specific” activations arecalculated from the frame immediately following the activation pulse andthe number of “non-specific” activations from subsequent imaging framesin the imaging cycle. Assuming that the probability of “non-specific”activations is constant across all frames, it could be determined thenumber of “actual specific” activations by subtracting the “non-specificactivation” number from the “apparent specific” activation number. Wethen used these numbers to statistically subtract crosstalk due to“non-specific” activations in an unbiased way as previously described(Dani et al., 2010).

Additionally, the position lists can be used to construct discretelocalization images, such that each pixel has a value equal to thenumber of localizations falling within the pixel area, as a way ofillustrative-non limitative example the pixel size is ≧the locationaccuracy, in a more preferred embodiment the pixel size is 10 nm. Fromthe localization images, density maps may be obtained by 2-dimensionalconvolution with a square kernel, as a way of illustrative-nonlimitative example, preferably ≧1×1 pixels², more preferably 5×5pixels², although the kernel can have different shapes. A constantthreshold may be used to digitize the density maps into binary images,such that pixels have a value of 1 where the density is larger than thethreshold value and a value of 0 elsewhere. Localizations falling onzero-valued pixels of the binary images (low-density areas) may bediscarded from further analysis. Connected components of the binaryimage, composed by adjacent non-zero pixels (4-connected neighborhood),are sequentially singled out and analyzed. Localization coordinateswithin each connected component can be grouped by means of adistance-based clustering algorithm. Initialization values for thenumber of clusters and the relative centroid coordinates can be obtainedfrom local maxima of the density map within the connected region,calculated by means of a peak finding routine. Localizations may beassociated to clusters based on their proximity to cluster centroids.New cluster centroid coordinates can be iteratively calculated as theaverage of localization coordinates belonging to the same cluster. Theprocedure was iterated until convergence of the sum of the squareddistances between localizations and the associated cluster and providedcluster centroid positions and number of localizations per cluster.Cluster sizes can be calculated as the standard deviation oflocalization coordinates from the relative cluster centroid.

In an embodiment, a super resolution image is rendered from the list oflocations (x,y) determined as the coordinates in the sample where anoptical emission of a photoswitchable fluorophore adapted to emit lightat a wavelength λ₂ is present. In an example, peaks in single-moleculeimage are identified wherein only values over a predetermined thresholdvalue are taken into account. The relevant values, those values over thethreshold value, are fit to a simple Gaussian to determine the x and ypositions over the image. The x and y position over the image can becorrelated to the physical x and y coordinates over the sample forinstance once the limits of the image over the sample are known. Then,the set of locations (x,y) may be provided as a list.

A further procedure uses data in a form of a list of coordinates (x, y),each coordinate (x, y) corresponding to one location of aphotoswitchable fluorophore.

Departing from the information having the location (x, y) of thefluorophores obtained from the image or images, in an embodiment of theinvention clutches and relevant parameters on said clutches is provided.

In a first step, a density image of resolution lower than or equal tothe rendered high resolution image used for the determination of thelocations (x, y) and representing the same area as said rendered highresolution image is provided wherein each pixel of the density image hasa value proportional to the number of locations of the location listfalling within the area represented by said pixel. In particular, thevalue is taken as the number of localizations falling within the pixelarea represented by the pixel.

In a second step, a binary image representing the same area than thedensity image comprising zero value pixels if the corresponding valuerepresented by the density image in the same location is lower than apredefined threshold; and, nonzero if said value is higher, is provided.Zero and nonzero values (for example 1), are examples of binary valuesrepresenting two different levels. A first level corresponding to pixelvalues under the threshold value and a second level corresponding topixel values equal or over the threshold value.

Regions of pixels corresponding to the second level comprise clutches,which are shows as clusters of pixels. A third step identifies connectedregions of pixels representing values higher than the predefinedthreshold, that is, the binary value representing the second level.

In a fourth step, the localization of clutches is identified from thebinary image and the list of localizations. For each connected region,the localization coordinates falling within said connected region isgrouped according to a distance-based criterion. Each group of locationsis deemed to belong to the same clutch.

The position of the clutch is taken as the centroid position of thelocalization coordinates associated with said clutch.

The fourth step provides a list of the position of clutches calculatedas disclosed. Once the position of the clutches being in each region,the number of clutches per region, the density calculated using adistance-based criterion and other statistical values may be used asmeasurements parameters for the determination of criteria that allowsdiscerning if a cell is in an open chromatin state or in a closechromatin state according to particular embodiments of the invention.

Thus, in an embodiment, a density image of resolution lower than orequal to the rendered high resolution image and representing the samearea than said rendered high resolution image is provided wherein eachpixel of the density image has a value proportional to the number oflocations of the list of location coordinates falling within the arearepresented by said pixel,

a binary image representing the same area than the density imagecomprising zero value pixels if the corresponding value represented bythe density image in the same location is lower than a predefinedthreshold; and, nonzero if said value is higher, is provided,

identifying connected regions of pixels representing values higher thanthe predefined threshold,

for each connected region, providing a list of clutch positions bygrouping the localization coordinates within said connected regionaccording to a distance-based criterion being the position of the clutchthe centroid position of the localization coordinates associated withsaid clutch.

In a preferred embodiment, the method comprises identifying connectedregions of nonzero pixels.

In a preferred embodiment, the size of nucleosomal clutch is calculatedas a measure of the spreading of the positions of all the localizationcoordinates associated with said clutch and/or the number of nucleosomeswithin said clutch.

In another preferred embodiment, the density of clutches within aconnected region is calculated as the number of nucleosomes per clutchdivided by the area occupied by said clutches.

The method of the invention further comprises step e) comparing dataobtained in step d) with a corresponding reference value to obtain ascore based on size of nucleosomal clutches, nucleosomal density and/ornumber of nucleosomes per nucleosomal clutch.

“Reference value”, as used herein relates to a laboratory value used asa reference for the values/data obtained from samples. The referencevalue (or reference level) can be an absolute value, a relative value, avalue which has an upper and/or lower limit, a series of values, anaverage value, a median, a mean value, or a value expressed by referenceto a control or reference value. A reference value can be based on thevalue obtained from an individual sample, such as, for example, a valueobtained from a sample of study but obtained at a previous point intime. The reference value can be based on a high number of samples, suchas the values obtained in a population of samples. In order to detect acell in an open chromatin state, the reference value can be based on theclutches area, number of nucleosomes per nucleosomal clutch, ornucleosome density of clutches from a cell in a close chromatin state,by way of illustrative non-limitative example from a non-cancer cell, aterminally differentiated cell or from a cell wherein the machinery oftranscription is inactive.

In another preferred embodiment, the reference value can be based on theclutch area, number of nucleosomes per nucleosomal clutch or nucleosomedensity of clutches from cells with an open chromatin state oralternatively with a more open chromatin state, by way of illustrative,non-limitative example highly transcriptionally activated cells, highlypluripotent cell, ESCs and iPSCs. Cells with a more open chromatin statemay correspond to cells with higher grade of pluripotency. The grade ofpluripotency in a cell can be determined, for example, with a gene cardtechnology (Bock et al, 2011).

In another preferred embodiment, in order to detect a cell in a closechromatin state, the reference value is based on the clutch area, numberof nucleosomes per nucleosomal clutch or nucleosome density of clutchesfrom cell kwnon to be I a close chromatin state.

In a preferred embodiment, the reference value for discriminating amongdifferent cell types, based on the number of nucleosomes per clutch byway of illustrative non-limitative example is <=5 nucleosomes perclutch. In another preferred embodiment, the reference value fordiscriminating different among different cell types, based on thedensity of nucleosomes per clutch by way of illustrative non-limitativeexample is <=0.005 nucleosomes/nm².

Once the reference value has been established, the size of nucleosomalclutches, nucleosomal density and/or number of nucleosomes pernucleosomal clutch is compared with the reference value. As aconsequence of this comparison the size of nucleosomal clutches,nucleosomal density and/or number of nucleosomes per nucleosomal clutchcan be “greater than” or “bigger than” or “more that”; “less than” or“smaller than” or “equal to” the corresponding reference value.

In the context of the present invention, the size of nuclesomalclutches, the nucleosomal density or the number of nucleosomes pernucleosomal clutches are “greater than or more than or bigger than” thecorresponding reference value, when the size of nuclesomal clutches, thenucleosomal density or the number of nucleosomes per nucleosomalclutches is by way of illustrative, non-limitative example, at least1.1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more whencompared with the reference value for said marker. On the other hand,the size of nuclesomal clutches, the nucleosomal density or the numberof nucleosomes per nucleosomal clutches are “lower than or smaller than”the corresponding reference value, when the size of nuclesomal clutches,the nucleosomal density or the number of nucleosomes per nucleosomaldecreases by way of illustrative, non-limitative example, at least 5%,10%, 25%, 50%, 75%, or even 100%.

According to the invention, if the cell comprises smaller clutches, lessdensely compacted nucleosomes or less nucleosomes per clutches comparedto the corresponding reference value is indicative that said cell is inan open chromatin state.

According to the invention if the cell comprises bigger clutches, moredensely compacted nucleosomes or more nucleosomes per clutches comparedto the corresponding reference value is indicative that said cell is ina close chromatin state

In another preferred embodiment, the method for detecting the chromatinstate of a cell further comprises detecting the RNA polymerase IIassociation to the nucleosome.

According to this aspect of the invention, if the RNA polymerase II ismore associated to the nucleosome is indicative that said cell is in anopen chromatin state.

“RNA polymerase II”, as used herein, relates to an enzyme that catalyzesthe transcription of DNA to synthesize precursors of mRNA and most snRNAand microRNA.

In a preferred embodiment, the RNA pol II subunit B1 is detected. Thesequence of RNA pol II subunit B1 in humans corresponds to the sequenceP24928 in the Uniprot database 3 Sep. 2014.

In another aspect, the invention further comprises detecting the linkerhistone H1.

“Histone H1”, as used herein relates to a protein involved with thepacking of the “beads on a string” sub-structures into a high orderstructure. The sequence of RNA H1 in humans corresponds to the sequenceQ02539 in the Uniprot database 3 Sep. 2014.

According to this aspect of the invention, if the histone H1 is moreassociated to the nucleosome is indicative that said cell is in a closechromatin state.

The association of RNA polymerase II or H1 to the nucleosome can bedetected by any method known in the art. In a preferred embodiment, theassociation is detected by multicolor super resolution imaging asdescribed in Bates et al., 2007.

Method for Isolating a Cell in an Open or Close Chromatin State

In another aspect, the invention relates to a method for isolating acell in an open chromatin state comprising

-   -   a) detecting the chromatin state of a cell by a method according        to the invention, and    -   b) isolating a cell having smaller clutches, less densely        compacted nucleosomes or less nucleosomes per clutches.

In a preferred embodiment the cell in an open chromatin state isselected from the group consisting of transcriptionally active cells,pluripotent cells, cancer cells and drug perturbed cells. In a morepreferred embodiment, the cell in an open chromatin state is apluripotent cell. In another aspect, the invention relates to a methodfor isolating a cell in a close chromatin state comprising

-   -   a) detecting the chromatin state of the cell by a method        according to the method of the invention, and    -   b) isolating a cell having bigger clutches, more densely        compacted nucleosomes or more nucleosomes per clutches.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

Kit of the Invention

In another aspect, the invention relates to a kit comprising a firstantibody capable of specifically binding to a histone protein and aphotoswitchable fluorophore linked-secondary antibody.

In the context of the present invention, “kit” is understood as aproduct containing the different reagents necessary for carrying out themethods of the invention packed so as to allow their transport andstorage. Additionally, the kits of the invention can containinstructions for the simultaneous, sequential or separate use of thedifferent components which are in the kit. Said instructions can be inthe form of printed material or in the form of an electronic supportcapable of storing instructions susceptible of being read or understood,such as, for example, electronic storage media (e.g. magnetic disks,tapes), or optical media (e.g. CD-ROM, DVD), or audio materials.Additionally or alternatively, the media can contain internet addressesthat provide said instruction.

In a preferred embodiment, the first antibody capable of specificallybinding to a histone protein and a photoswitchable fluorophorelinked-secondary antibody comprise at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90% or at least 100% of the total amount of reagentsforming the kit.

In a preferred embodiment, the histone protein is a core histoneprotein, more preferably histone H2B.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

Use of the Kit

In another aspect, the invention relates to the use of the kit accordingto the invention for detecting the chromatin state of a cell andisolating a cell in an open chromatin state or in a close chromatinstate.

In a preferred embodiment, the detection of the chromatin state of acell and the isolation of a cell in an open chromatin state or in aclose chromatin state is performed by a method of the invention.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

Device

According to another aspect of the invention, the method of the firstand second aspect of the invention may be carried out by means of adevice adapted to detect the chromatin state of a cell comprising:

-   -   a source of optical radiation adapted to emit light at a        wavelength λ₁ over an interrogation area adapted to receive a        biological sample,    -   an optical sensor sensible to a second wavelength λ₂ adapted to        measure the optical radiation at λ₂,    -   a control unit connected to the optical sensor and to the source        of optical radiation wherein said control unit is adapted to        carry out the method according to the invention.

The source of the optical radiation may be in the form of a lasersource. The interrogation area is the area where the sample is locatedand it is the area over which the optical sensor is aiming so that theimage taken by the optical sensor is the focused over the sample. Inparticular, the optical sensor is sensible to the second wavelength λ₂,that is, the wavelength of the radiation emitted by the photoswitchablefluorophores linked to the antibody:histone complex determining itslocation.

The control unit is configured to have the control over the source ofthe optical radiation and the optical sensor to allow recording imagesof samples when the photoswitchable fluorophores are excited.

In an embodiment wherein the device further comprises a source ofoptical radiation adapted to emit light at a wavelength λ₃ over aninterrogation area adapted to receive a biological sample, the controlunit is further adapted to carry out the method of the invention.

In an embodiment, the control unit is a programmable unit and is adaptedto execute a computer program. According to a further embodiment, thecontrol unit is an ASIC unit being programmed to carry out the controlover the source of the optical radiation and the optical sensor to allowrecording images of samples when the photoswitchable fluorophores areexcited.

In a further embodiment, the control unit is adapted to carry out apost-processing of the image for the assessment of parameters over asample. In a preferred example, the control unit is configured to carryout steps first, second, third and fourth for the calculation ofposition of clutches.

It is part of the invention a computer program configured to carry outany of the disclosed methods for processing images obtaining informationof clutches and the spatial distribution.

The invention will be described by way of the following examples whichare to be considered as merely illustrative and not limitative of thescope of the invention.

Material and Methods Cells.

Human fibroblasts (hFb) (BJ, Skin Fibroblast, American Type CultureCollection, ATCC® CRL-2522™) were cultured in DMEM supplemented with 10%FBS, 1× Non-essential AA, 1× GlutaMax and 1× penicillin/streptomycin.Human fibroblasts were treated with 300 nM of TSA (TricostatinA,Sigma-Aldrich) solution (TSA-hFbs) in complete growth medium for 24hours before imaging experiments. Human fibroblasts expressing theHistone H2B-SNAP fusion protein were obtained after drug selection ofnucleofected cells with the pSNAP-H2B Plasmid (N91795, New EnglandBioLabs) using the Amaxa Human Dermal Fibroblast Nucleofector Kit(Lonza, VPD-1001).

mESCs and mESCs^(Tcf3−/−) were previously described (Merrill et al.,2004). mESCs^(H1tkO) gift from Arthur I. Skoultchi (Fan et al., 2005)mESCs were cultured on gelatin in sLif medium composed by KO DMEMsupplemented with 15% FBS (Hyclone), 1× Non-Essential Amino acid, 1×GlutaMax (Invitrogen), 1× penicillin/streptomycin, 1×2-mercaptoethanoland 1,000 U/mL LIF ESGRO (Chemicon). mESCs were cultured also in 2iLifmedium composed by N2B27 medium supplemented with 3 μM CHIR99021, 1 μMPD0325901, 1,000 U/mL LIF and 1× penicillin/streptomycin for eightpassages before imaging experiments.

mNPCs were generated by culturing mESCs as cell aggregates with 5 μMretinoic acid (RA) as previously described (Bibel et al., 2007).Neuronal progenitors cells were fixed 2 days after plating dissociatedcellular aggregates.

In-Vitro Polynucleosome Arrays.

The regular 12-mer and 24-mer DNA templates (gift from S. Grigoryev,(Grigoryev et al., 2009)) were isolated from Escherichia Coli andreconstituted with native histone octamers from HeLa cells using the ‘Invitro Chromatin Assembly Kit’ (CA-vitro-003, DIAGENODE). Chromatin waspurified over a column of 4% agarose beads (cat#: A-1040-M, ABT, AgaroseBead Technologies) in a 0.5×20 cm Econo-Column (BioRad) and immediatelyused for experiments. To induce high compact folding before STORMimaging, the purified polynucleosomes were spotted on a coverglass andincubated over night at 4° C. in presence of 1 mM MgCl2 and 150 mM NaCl,then fixed with PFA 4% solution for 10 min at 4° C.

Mononucleosomes were reconstituted as described before (Workman andKingston, 1992). Briefly, naked 200 bp DNA was mixed with HeLa octamersin a 1:1 w/w ratio in a reconstitution mix with 10 mM Tris-HCl, pH 8, 20mM EDTA, 2M NaCl, 10 mM DTT, 2 mM 2-mercaptoethanol, 15 ng/μl BSA andleft in mini dialysis chamber in a floater for dialysis in a high saltconcentrated buffer for 2 h at 4° C. Then the samples were dialyzed over20 h at 4° C. continuously diluting the concentration of NaCl from 2M to0M. Mononucleosomes were collected from the mini dialysis chamber andcentrifuged. Then they were spotted on a coverglass and left at 4° C.overnight and finally fixed in PFA 4% for 10 minutes at 4° C. HeLa'soctamers were spotted on a coverglass and fixed in the same way afterovernight incubation at 4° C. without addition of salts.

12-polynucleosome arrays were prepared for EM according to standardprotocols (CA-vitro-003, DIAGENODE). Purified and undiluted samples wereapplied to air glow discharged continuous carbon (hydrophilic-negativelycharged surface), contrasted with Uranyl Formate and examined in aPhilips Biotwin microscope at 120 kV. Images were recorded on a KeenViewCCD camera (SIS Olympus) (Electron Microscopy Core Facility of EuropeanMolecular Biology Laboratory, EMBL Heidelberg).

Human Induced Pluripotent Stem Cells Generation and Characterization.

Integration-free hiPSCs were generated as described previously (Okita etal., 2011). Briefly a combination of episomal vectors encoding forOCT3/4-shp53, SOX2, KLF4 and L-MYC (Addgene, #27077, #27078, #27080) wasnucleofected in human skin. Fibroblast cells (BJ, American Type CultureCollection, ATCC® CRL-2522™) using the Amaxa Human Dermal FibroblastNucleofector Kit (Lonza, VPD-1001). Normal fibroblast medium (DMEMsupplemented with 10% FBS, 1× GlutaMax and 1× penicillin/streptomycin)was changed every day. On day 7, the nucleofected fibroblasts werereseeded onto a monolayer of feeders cells and on day 8 the normalmedium was changed to hiPSC medium (DMEM/F12, 20% KO-SR, 1× MinimumNon-Essential Amino acid, 1× GlutaMax (Invitrogen), 1×penicillin/streptomycin, 1000×2-Mercaptoethanol, supplemented with 10ng/mL fresh basic FGF just before feeding the cells). Medium was changedevery day the first week and then every 2 days. hiPSC colonies appeared˜20 days after nucleofection. 20 clones were picked and plated on humanfeeders adding ROCK inhibitor (Y27632) at 10 μM to the medium. Aftersome passages cells were collected using trypsin (0.05%) and plated onmatrigel coated plates. 5 different clones (#6, #8, #13, #16 and #20)were finally cultured and characterized.

hiPSC clones were plated on feeders and cultured in hiPSC medium. hiPSCsplated on matrigel were cultured with the MEF-conditioned hiPSC medium.

Alkaline Phosphatase.

The staining was carried out on cells fixed in 10% Neutral FormalinBuffer for 15 min at 4° C., and washed three times with distilled water.The samples were then incubated for 45 min at room temperature in 2 mlof the staining solution prepared as it follows: 0.005 g Naphthol ASMX-PO4 (Sigma, N5000), 0.03 g Red Violet LB salt (Sigma, F1625), 200 mlN,N-Dimethylformamide (DMF, Fischer Scientific, D1191), 25 ml ofTris-HCl (MW=157.6, pH 8.3, 0.2M), and 25 ml of distilled water. Thealkaline-phosphatase-positive cells showed a red color and were visibleunder phase-contrast microscopy.

Immunostaining of Stem Cell Markers.

The staining was carried out on cells fixed with 4% PFA for 15 min atroom temperature and permeabilized with 0.1% Triton X-100 (Sigma) in PBSfor 10 min. Samples were incubated in blocking buffer containing 10% BSA(Sigma) in PBS for 1 h and then where left overnight at 4° C. withprimary antibodies in solution with blocking buffer.

Primary antibodies used were: mouse monoclonal anti-Human SSEA-4 cloneMC-813-70 (STEMCELL technologies) diluted 1:50; mouse monoclonalanti-Human TRA1-60 clone TRA1-60R (STEMCELL technologies) diluted 1:50,mouse monoclonal anti-Oct3/4 (Santa Cruz Biotechnologies, sc-5279)diluted 1:100, rabbit polyclonal anti-Sox2 (SIGMA, s9072) diluted 1:200;rabbit polyclonal anti-Nanog (Abcam, ab21624) diluted 1:100. For eachprimary antibody a respective secondary antibodies conjugated to AlexaFluor (Invitrogen), was used for 40 min at room temperature diluted1:1000 in blocking buffer. The cells were then counterstained with DAPI(Vector Laboratories).

Embryoid Bodies (EBs) Formation.

The cells were harvested by trypsinisation and seeded in 96 well plateswith V-bottom (Corning Costar) in hiPSC medium supplemented with 10ng/ml bFGF and 10 μM ROCK inhibitor (Y27632). 48 h later the EBs wereremoved from the V-bottom well plates and transferred to 10 cm2 lowattachment dishes in hiPSC medium. After 24 h formed EBs were divided inthree parts for in vitro differentiation to meso-endo-ecto-lineages.

For differentiation to endoderm and mesoderm, EBs were propagated for 3more days in suspension with EB medium (KO DMEM, 10% FBS (Hyclone), 1×GlutaMax (Invitrogen), 1× penicillin/streptomycin) before being platedon gelatine coated plates in EB medium. The medium was changed every 2-3days until 15 days when samples were fixed and processed forimmuno-fluorescence staining. For mesoderm differentiation, the mediumwas supplied with 0.5 mM ascorbic acid. For immuno-staining, rabbitpolyclonal anti-Alpha Actin-Smooth Muscle (ThermoScientific, #RB-9010),1:100 dilution and rabbit polyclonal Anti-FOXA2 antibody (Abcam,ab40874), 1:500 dilution were used.

For differentiation to ectoderm, the EBs were propagated for 3additional days in suspension with N2B27 media (50% Neurobasal medium,50% DMEM/F12 media, 1× GlutaMax (Invitrogen), 1×penicillin/streptomycin) supplemented with 10 ng/mL bFGF, 20 ng/mL EGFand 1,000 U/mL LIF and then for 4 more days with the addition of 1 μM RAto the medium. Then EBs were collected, washed and dissociated byincubating with trypsin (0.25) for 10 min at room temperature, pipettingup and down. Cells were then collected and plated into matrigel-coatedplates in N2B27 supplemented with 10 ng/mL bFGF and 20 ng/mL EGF. 24 hlater the medium was changed to N2B27 alone and cells were maintained inculture for 20 days, until neuronal connection was seen in the dish.Cells were fixed and stained as explained in previous sections withmouse monoclonal anti-beta III Tubulin, TU-20 (Abcam, ab7751), 1:500dilution.

Teratoma Production.

Cells were collected, resuspended in matrigel and intratesticularinjected in SCID mice. After 7 weeks, formed teratomas were surgicallydissected, fixed, embedded, sectioned and stained with hematoxylin andeosin.

TaqMan hPSC Scorecard Panel.

hiPSCs, previously grown for one passage on matrigel without feeders,were collected and RNA extracted with the RNeasy Mini Kit (Quiagen),according to manufacturer instructions. Total RNA was treated with DNase(Quiagen) to prevent DNA Contamination. RNA integrity was controlled bybioanalyzer instrument. High Capacity cDNA Reverse Transcription(Invitrogen) was used to prepare cDNA according to TaqMan hPSC ScorecardPanel Workflow. qRT-PCR using the TaqMan hPSC Scorecard Panel wasprepared according to manufacturer instruction and run in Viia 7Real-Time PCR System. Raw Data were analyzed using the web-based hPSCScorecard™ Analysis Softwarev1.2, available atlifetechnologies.com/scorecardsoftware.

Immuno-Staining for STORM.

For the imaging experiments, cells were plated on S-well Lab-tek 1coverglass chamber (Nunc) at a seeding density of 20,000-50,000 cellsper well, fixed and permeabilized with Methanol-Ethanol (1:1) solutionat −20° C. for 6 min or fixed with PFA 4% in PBS for 10 min and thenpermeabilized with 0.1% v/v Triton X-100 (SIGMA) in PBS for 10 min atroom temperature. As the distribution of H2B was independent of thefixation and permeabilization protocols, Methanol-Ethanol (1:1) waspreferred to minimize the handling of the sample. After 1 h incubationat room temperature with blocking buffer containing 10% (wt/vol) BSA(Sigma) in PBS, samples were incubated overnight with the primaryantibody diluted 1:50 in blocking buffer and then for 40 min with theappropriate dilution of dye-labeled secondary antibodies. Repeatedwashing were done at every step. Primary antibodies used forimmunostaining experiments were: rabbit polyclonal anti-H2B (Abcam,abcam 1790); mouse monoclonal anti-Histone H1 Antibody, clone AE-4 (MerkMillipore, 05-457); rabbit polyclonal anti-SNAP-H2B (New EnglandBiolabs, P9310S); mouse monoclonal anti-Histone H2B, clone 5HH2-2A8(Merk Millipore, 05-1352); rabbit polyclonal anti-Acetyl-histone H3(Merk Millipore, 06-599); mouse monoclonal anti-RNA Polymerase II, cloneH5-phosphoserine 2 version of pol II (Covance, MMS-129R); mousemonoclonal anti-RNA Polymerase II, clone H14-phosphoserine 5 version ofpol II (Covance, MMS-134R); goat anti-GFP-Alexa Fluor 647 nanobody (giftfrom Jonas Ries), 1:1000 dilution.

Secondary antibodies used were donkey-anti mouse and donkey-anti rabbit.Secondary antibodies were all from Jackson ImmunoResearch. For STORMimaging, the secondary antibodies were labeled in-house with differentcombinations of pairs of activator/reporter dyes, as previouslydescribed (Bates et al., 2007). Briefly, the dyes were purchased as NHSester derivatives: Alexa Fluor 405 Carboxylic Acid Succinimidyl Ester(Invitrogen), Cy3 mono-Reactive Dye Pack (GE HealthCare), and AlexaFluor 647.

Carboxylic Acid succinimidyl Ester (Invitrogen). Antibody labelingreactions were performed by incubating for 40 min at room temperature amixture containing the secondary antibody, NaHCO3, and the appropriatepair of activator/reporter dyes diluted in DMSO. Purification of labeledantibodies was performed using NAPS Columns (GE HealthCare). The dye toantibody ratio was quantified using Nanodrop and only antibodies with acomposition of 3-4 Alexa Fluor 405 and 0.9-1.2 Alexa Fluor 647 perantibody were used for imaging.

For all H2B quantification experiments, primary rabbit polyclonalanti-H2B (ab1790) and the secondary donkey anti-rabbit labeled withAlexa Fluor 405-Alexa Fluor 647 (Invitrogen) pair dyes were used afterin vitro characterization using mononucleosome, 12- and 24-nucleosomearray labeling.

hiPSCs grown on feeder layers were co-stained for OCt3/4 (sc-5279) andH2B (ab1790), only the cells positive for the pluripotency marker werethen STORM imaged for H2B.

STORM Imaging.

STORM imaging was carried out with a commercial STORM microscope systemfrom Nikon Instruments (NSTORM). Laser light at 647 nm was used forexciting Alexa Fluor 647 (Invitrogen) and switching it to the darkstate, and laser light at 405 nm was used for reactivating the AlexaFluor 647 (Invitrogen) fluorescence via an activator dye (Alexa Fluor405)-facilitated manner. An imaging cycle was used in which one framebelonging to the activating light pulse (405 nm) was alternated withthree frames belonging to the imaging light pulse (647 nm). Dual colorimaging was performed with two sets of secondary antibodies labeled withthe same reporter dye (Alexa Fluor 647) but two different activator dyes(Alexa Fluor 405 and Cy3) (Bates et al., 2007). In addition to the 405nm laserlight, an additional imaging cycle with 561 nm laser light asthe activating light pulse was used for reactivating Alexa Fluor 647linked to the second activator dye (Cy3). The emitted light from AlexaFluor 647 was collected by an oil immersion 100× objective with 1.49 NA,filtered by an emission filter (ET705/72m) and imaged onto an electronmultiplying charge coupled device (EMCCD) (Andor Technology) camera at aframe rate of 15 ms per frame. For all single color and in vitro H2Bimaging experiments, identical ‘excitation-switching off-reactivation’scheme was used by gradually increasing the 405 nm laser power in asigmoidal manner starting with 0.5 μW at frame 800 and ending with 2000μW at frame 44800 according to Table I. Up to frame 800, the 405 nmlaser power was set to zero. When the final power of 2000 μW wasreached, this power was kept until the fluorophores were exhaustivelyimaged and photobleached. Imaging was done using a previously describedimaging buffer [Cysteamine MEA (SigmaAldrich, #30070-50G), GloxSolution: 0.5 mgmL⁻¹ glucose oxidase, 40 mgmL⁻¹ catalase (all Sigma),10% Glucose in PBS](Bates et al., 2007).

STORM Data Analysis.

STORM images were analyzed and rendered as previously described (Bateset al., 2007; Huang et al., 2008a; Huang et al., 2008b), usingcustom-written software (Insight3, kindly provided by Bo Huang,University of California, San Francisco). Briefly, peaks insingle-molecule images were identified based on a threshold and fit to asimple Gaussian to determine the x and y positions. The final imageswere rendered by representing each x-y position (localization) as aGaussian with a width that corresponds to the determined localizationprecision (9 nm). Sample drift during acquisition was calculated andsubtracted by reconstructing STORM images from subsets of frames(typically 500-1000 frames, for which drift was assumed to be small) andcorrelating these images to a reference frame (typically one that isreconstructed at the initial time segment). For multicolor images, eachpeak was color coded based on whether the emission was recordedimmediately after 405 nm or 532 nm activation cycle. The peaks comingfrom a frame not belonging to the one right after an activation framewere coded as “non-specific”. A crosstalk algorithm as describedpreviously was applied to correct for non-specific activations by theimaging laser (Dani et al., 2010). Briefly, the number of “apparentspecific” activations were calculated from the frame immediatelyfollowing the activation pulse and the number of “non-specific”activations from subsequent imaging frames in the imaging cycle.Assuming that the probability of “non-specific” activations is constantacross all frames, we could then determine the number of “actualspecific” activations by subtracting the “non-specific activation”number from the “apparent specific” activation number. We then usedthese numbers to statistically subtract crosstalk due to “non-specific”activations in an unbiased way as previously described (Dani et al.,2010).

Image Analysis and Cluster Quantification.

STORM data consisting in (x,y) localization lists were used to constructdiscrete localization images, such that each pixel has a value equal tothe number of localizations falling within the pixel area (pixel size=10nm). From the localization images, density maps were obtained by2-dimensional convolution with a square kernel (5×5 pixels²). A constantthreshold was used to digitize the density maps into binary images, suchthat pixels have a value of 1 where the density is larger than thethreshold value and a value of 0 elsewhere. For the determination of thethreshold value, unlabeled samples were imaged. The images were analyzedas described, and digitized with increasing threshold values. For eachthreshold value, the ratio of nonzero to zero pixels was calculated. Thethreshold value (0.002 nm⁻²) giving a ratio <2×10⁻⁴ was used for imageanalysis. Localizations falling on zero-valued pixels of the binaryimages (low-density areas) were discarded from further analysis. For ourthreshold setting, the number of discarded localizations typicallycorresponded to <5% of the total number of localization within a nuclearregion.

Connected components of the binary image, composed by adjacent non-zeropixels (4-connected neighborhood), were sequentially singled out andanalyzed. Localization coordinates within each connected component weregrouped by means of a distance-based clustering algorithm.Initialization values for the number of clusters and the relativecentroid coordinates were obtained from local maxima of the density mapwithin the connected region, calculated by means of a peak findingroutine. Localizations were associated to clusters based on theirproximity to cluster centroids. New cluster centroid coordinates wereiteratively calculated as the average of localization coordinatesbelonging to the same cluster. The procedure was iterated untilconvergence of the sum of the squared distances between localizationsand the associated cluster and provided cluster centroid positions andnumber of localizations per cluster. Cluster sizes were calculated asthe standard deviation of localization coordinates from the relativecluster centroid.

In order to further check the effect of the threshold on thequantification, a subset of data (hFbs, TSA-hFbs, mononucleosomes, 12-and 24-nucleosome array) was analyzed by applying different thresholdvalues, ranging from 8·10⁻⁴ to 0.004 nm². All the investigated datashowed similar linear dependence of the median number of localizationsper cluster versus the threshold value.

Analyses were performed by means of custom code written in Matlab.

Simulations of Synthetic Images.

Three-dimensional nucleosomes sequences were simulated assumingnucleosomes as impenetrable spheres (r=10 nm) arranged in spaceaccording to a Gaussian chain model. Inter-nucleosomes end-to-enddistances were calculated by conversion of DNA linker lengths accordingto worm like chain (WLC) model for a polymer with a persistence lengthof 150 bp.

It has been assumed that at full DNA occupancy (75% of DNA lengthcovered by nucleosomes) nucleosomes have 146 bp DNA wrapped around themand are uniformly spaced by linker DNA fragments of 50 bp (Kornberg andLorch, 1999).

For comparison with the experimental data, distributions of linker DNAlengths were modified on the basis of two different models. In the firstmodel (NR-model), we considered the possibility that nucleosomes can berandomly removed with a finite probability p. The removal of anucleosome results in increase in the DNA linker length betweenneighboring nucleosomes, caused by DNA unwrapping (146 bp). For thismodel, the DNA occupancy (OCC_(DNA)) depends on the nucleosome removalpercentage p as:

$\begin{matrix}{{OCC}_{DNA} = {{100\%} - {\lbrack {p + {( {{100\%} - p} )\frac{50\; {bp}}{( {50 + 146} ){bp}}}} \rbrack.}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

In the linker length (LL) model, we assume that nucleosomes are spacedby linker-DNA lengths distributed according to a normal distributionwith average length l_(average), so that the DNA occupancy is a functionof l_(average):

$\begin{matrix}{{OCC}_{DNA} = {\frac{lbp}{( {l + 146} ){bp}}.}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

Simulations were carried out for several nucleosome removal percentages(from 0 to 95%) and average linker length l_(average) (from 50 bp to˜3000 bp). For each parameter value, thousands of simulation weregenerated and analyzed.

In order to obtain synthetic STORM images of nucleosomes configuration,for each simulated nucleosome a number of localizations was randomlydrawn from the distribution obtained from STORM images ofmononucleosomes in vitro. To take into account the different efficiencyin detecting localizations at various distances from the focal plane (zaxis), the number of localizations was scaled with a z-dependent factorobtained by an independent calibration. This calibration consisted ofrepeatedly imaging the same sample at defined distances from the focalplane. The sample was moved by means of a piezoelectric stage.Quantification of the number of localizations versus the sample distanceprovided the z-dependent correction factor. Localizations were thenrandomly placed around the nucleosome centroid position according to a2-d Gaussian distribution with standard deviation equal to the oneobtained from STORM images of mononucleosomes in vitro.

The localization coordinates were then analyzed in the same way as theregular STORM images and the number of localization per cluster, clusterarea and nearest neighbor distance were quantified.

Example 1 Nucleosomes in Interphase Nuclei of Human Somatic Cells areOrganized in Discrete Nanodomains

To reveal the organization of chromatin at nanoscale resolution, theinventors recorded STORM images of the core histone protein H2B ininterphase human fibroblast nuclei (hFb). An antibody that recognizesnative H2B was used. STORM images revealed a striking organization ofH2B inside the nucleus (FIG. 1A, left), which was not evident withconventional fluorescence microscopy (FIG. 7A). H2B appeared clusteredin discrete and spatially separated nanodomains (FIG. 1A, right zooms).The H2B nanodomain density (number of nanodomains per unit area) was˜25% higher in the nuclear periphery, where the heterochromatin isthought to be located, compared to the nuclear interior. Since H2B is acore histone of the nucleosome octamer, its localization should reflectthe arrangement of nucleosomes within the chromatin fiber. In accordancewith this idea, another core histone protein of the nucleosome octamer,H3, was found to be similarly clustered in discrete nanodomains (FIG.7B).

To rule out the possibility that the observed clustered distribution ofH2B was due to sample preparation or labeling methods used, theinventors performed a series of control experiments. First, they showedthat the clustered distribution of H2B was independent of the fixationand permeabilization protocols used (FIGS. 7C and 7D). Second, STORMimages contained discrete nanodomains when H2B was indirectly labeledusing an antibody against SNAP tag in cells stably expressing H2B-SNAP(FIG. 7E). Third, to rule out potential artifacts in H2B STORM imagesassociated with the large size of the antibody probe, hFbs weretransfected with a plasmid carrying H2B-GFP. hFbs showed varying levelsof GFP-H2B expression as could be observed from the intensity of the GFPsignal in the nucleus (FIG. 7F, H, J, L). Transfected cells wereimmunostained with either an anti-H2B antibody or an anti-GFP nanobody,which has a smaller size (nanobodies are 13 kDa as opposed to ˜150 kDasize of the antibodies). Along with STORM images of H2B, conventionalfluorescence images of GFP were recorded in these cells to assess thelevel of H2B-GFP expression. STORM images of low-GFP expressing cells(FIGS. 7F and J) immunostained with either antibodies (FIG. 7G) ornanobodies (FIG. 7K) contained discrete nanodomains. When GFP expressionlevel was high (FIGS. 7H and L), STORM images contained much more H2Blocalizations (>5-fold increase) leading to a more homogenous appearanceof H2B inside the nucleus with no evident organization in nanodomains,both for cells that were immunostained with antibodies and nanobodies(FIGS. 7I and M). Taken together, these controls indicate that the sizeof the H2B-antibody does not significantly affect the detected labelingdensity. Finally, labeling efficiency defects were also ruled out bycomputer simulations of nucleosome arrangements (FIGS. 12A and 12B).

They next aimed to analyze the nucleosome organization in cellsundergoing massive epigenome modifications and chromatin rearrangements.Thus, hFbs were treated with Trichostatin A (TSA) (TSA-hFb), a potentinhibitor of histone deacetylase enzyme, which is known to lead togenome-wide decondensation of chromatin inside the nucleus throughaccumulation of acetylation groups on histone tails (Toth et al., 2004).As expected, there was a large increase in H3 acetylation after TSAtreatment (FIG. 7N). In STORM images, TSA treatment resulted in visuallyevident changes in the nuclear distribution of H2B nanodomains (FIG. 1A,right). The nanodomains appeared dimmer and hence contained lesslocalizations. Furthermore, these dimmer nanodomains were also moredispersed within the nucleus (FIG. 1A, right zooms). The H2B nanodomaindensity was enhanced by ˜10% in the nuclear periphery of TSA-hFbscompared to the nuclear interior, although it was less dense than thenuclear periphery of untreated hFbs. Finally, not only the acetylated H3levels were increased in TSA-hFbs but also the distribution ofacetylated H3 was highly dispersed in the nuclei, mirroring the spatialre-distribution observed for the H2B nanodomains after TSA treatment(FIG. 7N). These changes overall indicated that nucleosomes undergospatial rearrangement in hFb nuclei upon chromatin decondensation.

To gain quantitative insight into the H2B nanodomains, the inventorsnext developed a cluster identification algorithm to group the detectedlocalizations in STORM images into nanodomains (FIG. 1B). Briefly,images were converted into a density map and a threshold was set tofilter out low-density regions containing isolated localizations thatwere likely due to background and noise. They applied a stringentthreshold setting and only <5% of the total number of localizationswithin a given nuclear area was discarded (FIG. 10). The remaininglocalizations were converted into a binary image and all the regionsthat contained localizations in this binary image were retained forfurther analysis. Within these regions a maximum likelihood algorithmwas used to group the localizations together based on their spatialproximity. The number of localizations per nanodomain, nanodomain areaand the nearest neighbor distances (nnd) between nanodomains were thenquantified (FIG. 1C). The quantitative analysis revealed that thedistributions of the number of localizations per nanodomain, nanodomainareas and nanodomain nnds were shifted to lower values in TSA-hFbscompared to hFbs (FIG. 1C). In control experiments, nanodomain areas ofhFbs were similar when H2B was labeled with an antibody (MeanArea±SEM=831±66 nm2) or GFP-nanobody (Mean Area±SEM=663±23 nm2)(p=0.1347) in hFbs transfected with H2B-GFP, indicating that the largesize of the antibody did not significantly affect the spatial resolutionof H2B STORM images. Overall, these quantitative results confirmed thatH2B nanodomains, and hence nucleosomes showed statistically significantspatial re-organization (p<10⁻³) after TSA treatment and chromatindecondensation.

Example 2 Nucleosomes in Interphase Nuclei of Mouse Embryonic Stem CellsForm Discrete Nanodomains, Whose Organization Correlates with GroundState Pluripotency

To assess the H2B organization of pluripotent stem cells, the inventorsnext imaged mouse embryonic stem cells (mESC) with STORM. mESCs wereinitially cultured in a medium containing serum and the Leukemiainhibitory factor (sLif) and H2B was labeled by immunofluorescence andimaged with STORM as before. STORM images of these mESCs showed twodifferent categories of nuclei. The first category, Type 1, displayednanodomains that appeared bright (i.e. contained a large number oflocalizations) similar to hFbs (FIG. 2A, yellow arrowheads). The secondcategory, Type 2, on the other hand, displayed an increased amount ofdimmer nanodomains (i.e. containing a small number of localizations) andthe nanodomains were more dispersed within the nucleus, similar toTSA-hFbs (FIG. 2B, cyan arrowheads). It has been reported that mESCscultured in sLif medium are heterogeneous and some cells are primed todifferentiation (Marks et al., 2012). The inventors thus hypothesizedthat the differences in Type 1 and Type 2 mESCs could be due todifferent levels of pluripotency. To test this hypothesis, Nanog, apluripotency marker, was imaged with conventional fluorescencemicroscopy and STORM images of H2B were recorded. As can be deduced fromthe density images (FIG. 8A), cells belonging to Type 1 expressed lowlevels of Nanog whereas those belonging to Type 2 expressed high levelsof Nanog. These results are consistent with the interpretation thatdifferences in H2B nanodomain organization of mESCs are correlated withdifferences in pluripotency. To further validate this observation, mESCswere next cultured in a medium containing 2i and Lif. The 2iLif mediumcontains inhibitors for the mitogen-activated protein kinase signalingand glycogen synthase kinase-3, which allow mESCs to be propagated intheir ground-state of pluripotency. Nanodomains of mESCs cultured in the2iLif medium were mostly dim and resembled the nanodomain organizationof more pluripotent Type 2 mESCs, further confirming the correlationbetween pluripotency and H2B nanodomain organization (FIGS. 2C and 2G).In contrast, when mESCs were differentiated into neural precursor cells(mNPCs) the H2B nanodomains became brighter, resembling the somaticphenotype of hFbs (FIGS. 2D and 2H).

The activation of Wnt/β-catenin signaling pathway controls mESCpluripotency and self-renewal (Kuhl and Kuhl, 2013). Tcf3 acts as a keyeffector of this pathway by repressing Wnt target genes. Its deletion inmESCs maintains the ground state of pluripotency. H2B nanodomains in theSTORM images of mESCs in which Tcf3 was knocked-out (mESCs^(Tcf3−/−))resembled those found in naïve pluripotent mESCs (Type 2 and 2iLif)(FIG. 2E). In ESCs^(Tcf3−/−) large epigenome modifications werepreviously seen as well as the increase of H3 acetylation (Lluis et al.,2011). Accordingly, there was increased level of acetylation in thesecells (FIG. 8B) with respect to Type 1 mESCs cultured in sLif (FIG. 8C).Interestingly, the more pluripotent mESCs cultured in 2iLif (FIG. 8D) aswell as the group of more pluripotent Type 2 mESCs cultured in sLif(FIG. 8E) also contained higher levels of H3 acetylation, while mNPCsshowed a lower level of H3 acetylation (FIG. 8F). Of note, the spatialdistribution of H3 acetylation resembled H2B nanodomain organization.Taken together, these results indicated that the ground state ofpluripotency in mESCs was associated with increased acetylation anddimmer H2B nanodomains, which were more dispersed inside the nuclearspace.

H1 is the linker histone that binds to the entry and exit sites of DNAthat is wrapped around the histone octamer, keeping the nucleosome inplace and leading to higher order compaction of the chromatin structure(Woodcock et al., 2006). Therefore, H1 is thought to play an importantrole in chromatin organization. For example, mESCs that lack three H1iso forms were shown to have chromatin structural changes such asreduced local chromatin compaction (Fan et al., 2005). To test whetherthe nanodomain organization of mESCs depended on H1, they imaged mutantmESCs carrying a deletion of three H1 isoforms (mESC^(H1tKO)). STORMimaging of H2B in these cells showed a large amount of dim nanodomains(FIG. 2F) having a similar organization to those observed in mESCscultured in 2iLif and in mESCs^(Tcf3−/−).

Quantification of the differences in nanodomain features among thevarious mESCs also confirmed that the number of localizations pernanodomain and nanodomain nnds were lower in ground-state mESCs withrespect to somatic mNPCs (FIG. 2I) (see also FIG. 3 and associated textbelow for further quantitative analysis).

Example 3 Nanodomains Contain a Discrete Number of Nucleosomes and theNucleosome Number Correlates with Cell Pluripotency

The inventors next aimed to further quantify the changes they observedin the number of localizations (and hence brightness) of nanodomains interms of the number of nucleosomes. There is not a one-to-onerelationship between the number of localizations in STORM images and thenumber of nucleosomes mainly for two reasons: i) the antibody epitopelabeling efficiency may not be 100%, ii) each fluorophore can undergomultiple photoswitching events, resulting in multiple localizationsarising from a single fluorophore. However, the epitope labelingefficiency of the H2B antibody should be comparable across the humancells (hFbs, TSA-hFbs) and likewise across the different mESCs analyzed.In addition, the antibodies used were always labeled with a similar dyecomposition (Extended Experimental Procedures) and each cell was imagedin the same way (Table I) to obtain comparable number of localizationsper antibody. Therefore, the number of nucleosomes should scale with thenumber of localizations. A similar approach has previously been used toquantify the receptor heterogeneity of synapses in brain slices (Dani etal., 2010).

TABLE I 405 nm laser power scheme used to activate fluorophores duringSTORM imaging 405 nm Laser Power (μW) Frame Number 0.5 800 3 7200 7 800022 10000 45 15200 78 20000 127 24000 200 28000 290 32000 500 36000 100040000 1630 42800 2000 44800

Nanodomains in any given nucleus contained a large distribution oflocalizations spanning two orders of magnitude (˜3 to 300) (FIGS. 1C and2I), indicating that these nanodomains comprised heterogeneous groupswith varying numbers of nucleosomes. The inventors will refer to theseheterogeneous nucleosome groups as “nucleosome clutches” in analogy to“egg clutches” and they will use the term “clutch size” interchangeablywith the number of nucleosomes per clutch. Despite this heterogeneity,the median number of localizations per clutch in individual cellscorrelated strongly with cell type and showed statistically significantdifferences between hFbs and TSA-hFbs as well as among the differentmESCs (FIGS. 3A and B). Control experiments showed that the mediannumber of localizations per clutch in hFbs was very similar when H3 waslabeled (Nlocalizations=24±2) instead of H2B (Nlocalizations=24±4) andunder different fixation and permeabilization conditions(Nlocalizations=24±4 for Ethanol/Methanol fixation, Nlocalizations=26±3for PFA fixation), excluding potential sample labeling artifacts andfurther validating our approach. Overall, the differences in the mediannumbers of localizations indicate that nucleosomes assembled into largerclutches in hFbs compared to TSA-hFbs (FIG. 3A). Similarly, nucleosomesformed larger clutches in differentiated mNPCs and less pluripotentmESCs (mESC cultured in sLif) compared to the more pluripotent mESCs(mESC cultured in 2iLif and mESC^(Tcf3−/−)) and mESC^(H1tKO) (FIG. 3B).

In order to relate the median number of localizations to the mediannumber of nucleosomes while taking into account the limitationsmentioned above, the inventors generated an in vitro calibration curve.To this end, single nucleosomes were assembled, spotted on coverglass invitro, labeled using the same cell immunostaining protocol and STORMimages were obtained using identical imaging conditions (Table I). Asexpected, the mononucleosome images resembled small clusters oflocalizations (FIG. 9A). The distribution of the number of localizationsper mononucleosome was quantified as before (FIG. 9B). Next,polynucleosome arrays were assembled, spotted on glass under high saltconditions to induce compaction, immunostained and imaged. The plasmidsused had a length and a specific DNA content allowing the assembly ofexactly 12- and 24-nucleosomes (Grigoryev et al., 2009). Electronmicroscopy images of the in vitro assembled polynucleosome arrays showedthat these structures had expected sizes for the number of nucleosomesthat were in the arrays (FIG. 9C). STORM images of the polynucleosomearrays also resembled small clusters (FIG. 9A) and the distribution ofthe number of localizations per polynucleosome array was quantified asbefore (FIG. 9B). The median number of localizations in the mono-, 12-and 24-nucleosome data was used to generate the calibration curve (FIG.3C).

The calibration curve was first validated by imaging nucleosomesassembled with circular DNA in vitro. The plasmid used had a DNA lengthallowing the assembly of ˜20 nucleosomes. The polynucleosomes assembledwith this plasmid were immunostained, imaged and the number oflocalizations per polynucleosome was quantified. The median number oflocalizations obtained was then interpolated from the calibration curveinto the median number of nucleosomes and corresponded to 19.5±2.0nucleosomes, confirming that the calibration curve was indeed accurate(FIG. 3C). Furthermore, to get an estimate of the number of antibodiespresent on each mononucleosome, single fluorophore-labeled secondaryantibodies were immobilized on coverglass, imaged with STORM (FIG. 9A)and the number of localizations per antibody was quantified as before(FIG. 9B). The median number of localizations per antibody was 9, whichis in agreement with previously measured photoswitching kinetics of theAlexa647 fluorophore (Nieuwenhuizen et al., 2013). This median numbercorresponded to 0.6 nucleosomes in the calibration curve (FIG. 3C,inset). Therefore, they calculated that on average 1.6 antibodies(1/0.6) were present on one mononucleosome.

The inventors next proceeded to convert the median number oflocalizations obtained from the in vivo STORM images (FIGS. 3A and 3B)to the median number of nucleosomes per clutch in the different celltypes (FIG. 3D). It is possible that antibody epitope binding efficiencymight be different between in vivo and in vitro conditions. Inparticular, due to nuclear crowding, antibody epitope binding efficiencymight be lower in vivo than it is in vitro, which would lead to anunderestimation of the number of nucleosomes per clutch. Nevertheless,this underestimation will not impact our conclusions regarding therelative relationship among the different cell types.

The median number of localizations in hFbs corresponded to a mediannumber of ˜8 nucleosomes per clutch whereas this number decreased to ˜2nucleosomes after TSA treatment (FIG. 3D, left). The mESCs cultured insLif medium contained a median number of ˜5 nucleosomes per clutch (FIG.3D, right). These cells comprised a rather heterogeneous populationconsisting of cells with a median of ≧5 nucleosomes and cells with amedian of <5 nucleosomes per clutch. The cells that contained ≧5nucleosomes per clutch were the same population as the Type 1 mESCs. Themedian number of nucleosomes per clutch increased to ˜6 in mNPCs (FIG.3D, right). The mESCs cultured in 2iLif and mESC^(Tcf3−/−) contained amedian number of ˜3 and ˜3.5 nucleosomes per clutch, respectively (FIG.3D, right). Finally, a median number of only ˜2 nucleosomes per clutchwere found in mESCs^(H1tKO) (FIG. 3D, right). While the variability inthe median number of nucleosomes per clutch was high among the differentnuclei analyzed for mNPCs and mESCs cultured in sLif, this number wasmore uniform among the different nuclei analyzed for 2iLif,ESCs^(Tcf3−/−) and mESCs^(H1tKO) (FIG. 3D, right). These results overallindicate that nucleosomes are assembled together in smaller clutches incells with pluripotent features and in increasing numbers in somaticdifferentiated cells. Furthermore, clutch size drastically changes uponchromatin decondensation after TSA treatment and upon differentiation ofmESCs into mNPCs.

To determine whether the observed changes in nucleosome clutchescorresponded to changes in the compaction of nucleosomes inside theclutches, the median nucleosome density was calculated by dividing themedian number of nucleosomes per clutch with the median area of theclutches. Indeed, the nucleosomes in hFbs were more densely compactedinside the clutches compared to TSA-hFbs (FIG. 3E). Nucleosome densitywas likewise higher for mNPCs and mESCs cultured in sLif with respect tomESCs cultured in 2iLif medium, mESCs^(Tcf3−/−) and mESCs^(H1tKO) (FIG.3F). These results show that nucleosomes are less densely compacted inpluripotent cells and nucleosome compaction increases upondifferentiation.

Example 4 Nucleosome Content Inside Clutches is Predictive of thePluripotency Grade of Human Induced Pluripotent Stem Cells

Given the correlation between clutch size and pluripotency level ofdifferent mESCs, the inventors next aimed to study whether theidentification of the number of nucleosomes per clutch could bepredictive of the pluripotency grade in hiPSC clones.

To this end, different hiPSC clones were generated from hFbs and werecharacterized using standard methods such as alkaline phosphatase (AP)staining, analysis of expression of stem cell genes, formation ofembryoid bodies and in vivo teratoma in immune-compromised mice. Basedon the results of this characterization, hiPSC clone 13 was the mostpluripotent since it was AP positive, expressed high levels of the stemcell markers TRA1-60, SSEA4, Oct4, Sox2 and Nanog, formed embryoidbodies, which differentiated in the three germ layers, and generatedlarge and fully differentiated teratomas in mice (FIG. 10A-D). Incontrast, the hiPSC clone 8 was the least pluripotent since, although itshowed expression of stem cell markers, the OCt4 expression level ofsingle cells was fourteen-fold lower compared to hiPSC clone 13, it didnot form the ectoderm layer from the embryoid bodies and it generatedvery small undifferentiated teratomas in vivo (FIG. 10A-D). To rank thepluripotency grade of all hiPSC clones in a more quantitative manner,they used a recently established gene card technology that compares theexpression level of stemness genes to well established standard hiPSCclones and gives a pluripotency score based on this comparison (Bock etal., 2011). The gene card results agreed with the classicalcharacterization, showing clone 8 as being the least and clone 13 asbeing the most pluripotent and allowed quantitative ranking of the restof the hiPSC clones in order of pluripotency grade (FIG. 10E).

In a double blind fashion, the median number of localizations wasquantified after STORM imaging of all the hiPSC clones (FIG. 4A), whichshowed statistically significant differences among the different clonesand a gradual increase of the median number of localizations passingfrom the more pluripotent hiPSCs clone, #13, to the less pluripotentone, #8. The calibration curve was used to deduce the median number anddensity of nucleosomes inside clutches in each hiPSC clone (FIGS. 4B and4C). There was a remarkable agreement between the pluripotency scoreobtained from the gene card and the clutch size (FIG. 4D), (correlationanalysis showed r=−0.94 indicating high level of anticorrelation, lownumber of nucleosomes per clutch for high pluripotency score and viceversa). Indeed, the more pluripotent hiPSC clone 13 had clutches with amedian number of only 1 nucleosome and low density (FIGS. 4B and C)while clutch size and density increased progressively with the decreasein pluripotency of the hiPSC clones.

Example 5 Larger Clutches have Higher Levels of H1 and Lower Levels ofRNA Polymerase II

The arrangement of nucleosomes in large and small clutches with higherand lower compaction respectively could potentially facilitate thebinding of transcription factors, polymerases and other proteins to theDNA, which should be more accessible in regions containing a smallernumber of nucleosomes. The compaction of the nucleosomes within theclutches, on the other hand, should be aided by the presence of linkerhistone protein H1, which is known to be involved in nucleosomecompaction as well as to be enriched in heterochromatin (Fan et al.,2005; Shen et al., 1995; Woodcock et al., 2006). Thus, to evaluatedifferences in the heterochromatin content and accessibility of RNAPolymerase II (PolII), multi-color super-resolution imaging of H2B withhistone H1 and of H2B with PolII was carried out. For H1, an antibodythat recognizes all of its isoforms was used. In the case of PolII, anantibody against phosphoserine 5 of the carboxiterminal domain of PolII(PolII11) was used to image both PolII at the initiation complex and theelongating PolII.

The inventors first recorded multi-color STORM images of H2B and H1 inhFbs and TSA-hFbs (FIGS. 5A and 5B), which significantly differ in themedian number of nucleosomes per clutch (FIG. 3D). H1 was more enrichedat the nuclear periphery of hFbs where heterochromatin is more abundant(Meister and Taddei, 2013) (FIG. 5A). A higher percentage of H2Bco-localized with H1 in hFbs (61±11%) compared to TSA-hFbs (42±6%)(p=0.028). They next counted the number of H1 localizations and comparedit to the number of H2B localizations inside the clutches (FIG. 5C). Forboth hFbs and TSA-hFbs the number of H1 localizations increased with thenumber of H2B localizations. Although they did not generate acorresponding calibration curve to convert from the number of H1localizations to the number of H1 histones, this result overall suggeststhat the number of H1 histones correlates with the number of nucleosomesinside the clutches.

In mESCs cultured in sLif around 54±2% of H2B colocalized with H1 andthe number of H1 localizations also increased with the number of H2Blocalizations (FIG. 11A). As expected, mESCs^(H1tKO) contained muchlower amount of H1 (FIG. 11B) and only around 35±4% of H2B colocalizedwith H1 (p=0.0057). Despite the low amount of H1 in these cells, thesame trend was observed in which the number of H1 localizations wasincreased in clutches with an increasing number of H2B localizations(FIG. 11A). These results overall indicate that H1-H2B colocalizationincreases in cells containing larger clutches and in addition theselarge clutches contain more H1 compared to the small ones.

Next, the inventors analyzed PolII and H2B multi-color STORM images ofhFbs and TSA-hFbs. In both cases, PolII was found interspersed with thenucleosome clutches (FIG. 5D and insets). Nearest neighbor distanceanalysis showed that the PolII-H2B distance peaked at around 40 nm (FIG.5E, top plot). They rationalized that the analyzed the number of H2Blocalizations within clutches as a function of the nearest neighbordistances between PolII and H2B. Even though hFbs (blue) and TSA-hFbs(red) have clutches with very different sizes, in both cases the nearestneighbor distances between PolII and H2B were shorter for smallerclutches, indicating that PolII was indeed closer to the smallerclutches with few nucleosomes (FIG. 5E, bottom plot). These resultsindicate that PolII can access small clutches, which likely form thechromatin fiber arrangement of transcribed chromatin regions.

Example 6 The DNA Fiber is not Fully Occupied with Nucleosomes

The organization of nucleosomes in discrete clutches that are separatedin space implies that nucleosome-depleted regions likely exist in thechromatin fiber. The inventors hypothesized that these regions might bethe result of two alternative mechanisms. First, removal of one or morenucleosomes in between nucleosome-rich regions can generate theclutch-like organization of nucleosomes observed in STORM images.Second, variations in the length of the linker-DNA between subsequentnucleosomes can generate nucleosome-depleted regions if the linker-DNAlength becomes larger than the spatial resolution.

To gain more insight into nucleosome occupancy in human fibroblastcells, they used coarse-grained computer simulations of nucleosomespatial arrangement, using a simple model with only few parameters.Nucleosomes were placed along the DNA fiber at regular intervals with146 bp of DNA wrapped around each nucleosome and with 50 bp oflinker-DNA separating them, which is the average linker-DNA lengthmeasured in previous studies, (FIG. 6A). They define this configurationas full occupancy of DNA, which in reality corresponds to ˜75% of DNAoccupied with nucleosomes after accounting for the 50 bp linker DNA.Final nucleosome positions were generated according to a Gaussian chainmodel (FIG. 6B). Inter-nucleosome distances (le-e) were calculated byconverting the linker-DNA length (50 bp) into an end-to-end distanceaccording to the worm-like-chain theory for a polymer with 150 bppersistence length, which is the experimentally determined persistencelength of double stranded DNA. The resulting DNA fiber configuration wasthen projected onto two-dimensional space (FIG. 6C).

To simulate nucleosome removal, the linker-DNA length was kept fixed at50 bp but nucleosomes were randomly removed with a probability rangingfrom p=0 to p=95% (NR model) (FIG. 6D). When a nucleosome was removed,the 146 bp DNA wrapped around this nucleosome unraveled and the twoneighboring nucleosomes became separated by 50+146+50 bp of DNA. In thismodel, a low probability of nucleosome removal led to high nucleosomeoccupancy of the DNA fiber and vice versa. The nucleosome occupancylevel was calculated according to Equation 1 shown before.

To simulate variations in linker-DNA length (1) between subsequentnucleosomes (LL Model) the 146 bp of DNA wrapped around nucleosomes wasmaintained, but the linker-DNA lengths (1) were extracted from normaldistributions with average linker-DNA lengths (laverage) ranging from 50bp to ˜3000 bp (FIG. 6E). Nucleosome occupancy in the LL model dependedon l_(average) with small l_(average) average leading to high occupancyand vice versa. The nucleosome occupancy level was calculated accordingto Equation 2 previously shown.

Finally, as a control, the potential effect of labeling efficiency wasalso simulated by decorating the nucleosomes with antibodiescorresponding to an average of 1.6 antibodies per nucleosome (in vitromeasured value, FIG. 3C, inset) and progressively decreasing thelabeling efficiency by randomly removing antibodies from thenucleosomes.

Synthetic STORM images of the nucleosomes along the DNA fiber atdifferent levels of nucleosome occupancy (FIG. 6F) or labelingefficiency (FIG. 12A) were generated by assigning to each nucleosome agiven number of localizations. The number of localizations was extractedfrom the experimental distribution of the in vitro mononucleosome (forNR and LL models) or secondary antibody data (for labeling efficiencysimulations) (FIG. 9B). The synthetic STORM images at differentnucleosome occupancy levels (FIG. 6F) showed striking resemblance to theexperimental images with nucleosomes grouped in clutches generating highand low density regions. The number of localizations, area and nearestneighbor distances of the nucleosome clutches in these synthetic STORMimages were determined using identical analysis parameters as before,including threshold. The median number of localizations, the mediannearest neighbor distances and the median area obtained from the NRmodel (filled squares and solid line) and the LL model (open circles anddashed line) were plotted as a function of nucleosome occupancy level(FIG. 6G). These data are the results of at least thousand simulationsfor each parameter value. Also shown are the corresponding median valuesobtained from the experimental data for hFbs (blue line) and TSA-hFbs(red line).

In the case of the number of localizations, both the NR and LL modelsintersected the experimental values at around 56% and 46% occupancy forthe hFbs and TSA-hFbs, respectively (FIG. 6G, top). Similarly, in thecase of the nearest neighbor distances, both the NR and LL modelsintersected the experimental values at around similar level of occupancyfor both cell types (57% hFbs and 43% TSA-hFbs) (FIG. 6G, middle). Inthe case of the nanodomain area, the NR model intersected theexperimental value for TSA-hFbs at similar level of occupancy (45%)whereas the LL model intersected the experimental value at a much loweroccupancy level (34%) (FIG. 6G, bottom). In the case of the hFbs, the NRmodel intersected the experimental value at a slightly higher occupancylevel than those obtained from the other two parameters (60%) whereasthe LL model intersected this value at a slightly lower occupancy level(52%) (FIG. 6G, bottom). Finally, in the case of labeling efficiencytests, the three measured experimental parameters (number oflocalizations, area and nearest neighbor distances) could not besimultaneously reproduced at any given labeling efficiency for hFbs andTSA-hFbs (FIG. 12B), indicating that poor labeling efficiency alonecannot explain the experimental observations.

Taken altogether, these results indicate that the nucleosome occupancyin TSA-hFbs is around 45% and nucleosome removal is likely the dominantmechanism to generate nucleosome poor regions along the DNA fiber sinceall three measured parameters of the experimental data can berecapitulated with this model. In hFbs, around 56% of the DNA fiber isoccupied with nucleosomes and likely both nucleosome removal andlinker-DNA length modifications play a role in generating thenucleosome-depleted regions. For these nucleosome occupancy levels (45%and 56%) the simulation results not only reproduced the median valuesobserved for the experimental data but also the full experimentaldistributions of the three parameters fit well to the simulateddistributions (FIG. 12C-E).

REFERENCES

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1. A method for detecting the chromatin state of a cell comprising a)contacting a sample containing cells with a first antibody capable ofspecifically binding to a histone protein, b) contacting theantibody:histone complex formed in step a) with a secondary antibodyhaving at least one photoswitchable fluorophore adapted to be opticallyexcited at a certain wavelength λ₂ and to emit light at a wavelength λ₂different from λ₂, c) recording a super resolution image of nucleosomeorganization by means of a sensor being sensitive at least to thewavelength of emission of the photoswitchable fluorophore by excitingthe sample with an optical radiation having a wavelength λ₂, d)correlating the image obtained in step c) with size of nucleosomalclutches, nucleosomal density and/or number of nucleosomes pernucleosomal clutches, and e) comparing data obtained in step d) with acorresponding reference value to obtain a score based on size ofnucleosomal clutches, nucleosomal density and/or number of nucleosomesper nucleosomal clutch, wherein if the cell comprises smaller clutches,less densely compacted nucleosomes or less nucleosomes per clutchescompared to the corresponding reference value then it is indicative thatsaid cell is in an open chromatin state and wherein if the cellcomprises bigger clutches, more densely compacted nucleosomes or morenucleosomes per clutches compared to the corresponding reference valuethen it is indicative that said cell is in a closed chromatin state. 2.The method according to claim wherein the cell in an open chromatinstate is selected from the group consisting of a transcriptionallyactive cell, a pluripotent stem cell, a cancer cell and a drug perturbedcell.
 3. The method according to claim 1, wherein the secondary antibodyfurther comprises a second fluorophore adapted to be optically excitedat a wavelength λ₃ and reactivate the first fluorophore by bringing itfrom its dark state back to its ground state, upon which the firstfluorophore can be excited again at its excitation wavelength λ₂ andemit light at its emission wavelength λ₂.
 4. The method according toclaim 3, wherein a plurality of super resolution images are taken bymeans of a sensor being sensitive at least to the wavelength of emissionof the second fluorophore λ₂ rendering a further super resolution imageby collecting the sensed light emissions recorded in the plurality ofimages.
 5. The method according to claim 4, wherein the power of theoptical radiation having a wavelength λ₂ is monotonically increased. 6.The method according to claim 4, wherein, before recording each superresolution image of the plurality of super resolution images, the sampleis excited one or more times with an optical radiation having awavelength λ₂ and subsequently excited one or more times with an opticalradiation having a wavelength λ₂.
 7. The method according to claim 4,wherein the super resolution image is rendered from a list of locations(x,y) determined as the coordinates in the sample where an opticalemission of a photoswitchable fluorophore adapted to emit light at awavelength λ₂ is present.
 8. The method according to claim 7, wherein adensity image of resolution lower than or equal to the rendered highresolution image and representing the same area as said rendered highresolution image is provided wherein each pixel of the density image hasa value proportional to the number of locations of the location listfalling within the area represented by said pixel, a binary imagerepresenting the same area as the density image comprising zero valuepixels if the corresponding value represented by the density image inthe same location is lower than a predefined threshold; and, nonzero ifsaid value is higher, is provided, identifying connected regions ofpixels representing values higher than the predefined threshold, foreach connected region, providing a list of clutch positions by groupingthe localization coordinates within said connected region according to adistance-based criterion being the position of the clutch the centroidposition of the localization coordinates associated with said clutch. 9.The method according to claim 8, wherein the size of each clutch iscalculated as a measure of the spreading of the positions of all thelocalization coordinates associated with said clutch and/or the numberof nucleosomes within said clutch.
 10. The method according to claim 8,wherein the density of nucleosomes within a clutch calculated as thenumber of nucleosomes within that clutch divided by the area occupied bysaid clutch.
 11. The method according to claim 1, wherein the histoneprotein is H2B.
 12. A method for isolating a cell in an open chromatinstate comprising a) detecting the chromatin state of a cell by a methodaccording to claim 1, and b) isolating a cell having smaller clutches,less densely compacted nucleosomes or less nucleosomes per clutches. 13.The method for isolating a cell in an open chromatin state according toclaim 12 wherein the cell in an open chromatin state is selected fromthe group consisting of transcriptionally active cell, pluripotent cell,cancer cell and drug-perturbed cell.
 14. The method for isolating a cellin a close chromatin state a) detecting the chromatin state of the cellby a method according to claim 1, and b) isolating a cell having biggerclutches, more densely compacted nucleosomes or more nucleosomes perclutches.
 15. A kit comprising a first antibody capable of specificallybinding to a histone protein and a photoswitchable fluorophorelinked-secondary antibody.
 16. The kit according to claim 15, whereinthe histone protein is histone H2B.
 17. Use of the kit according toclaim 15 for detecting the chromatins state of a cell and isolating acell in an open chromatin state or in a close chromatin state.
 18. Adevice adapted to detect the chromatin state of a cell comprising asource of optical radiation adapted to emit light at a wavelength, overan interrogation area adapted to receive a biological sample, an opticalsensor sensible to a second wavelength adapted to measure the opticalradiation at λ₂, a control unit connected to the optical sensor and tothe source of optical radiation wherein said control unit is adapted tocarry out the method according to claim
 1. 19. A device adapted todetect the chromatin state of a cell comprising a first source ofoptical radiation adapted to emit light at a wavelength λ₂ over aninterrogation area adapted to receive a biological sample, a furthersecond source of optical radiation adapted to emit light at a wavelengthλ₂ over an interrogation area adapted to receive a biological sample, anoptical sensor sensible to a second wavelength λ₂ adapted to measure theoptical radiation at λ₂, a control unit connected to the optical sensorand to the first and to the second source of optical radiation whereinsaid control unit is adapted to carry out the method according to claim3.